[0001] The invention relates generally to enzymatic activity involved in ENR-A, CBL, UROD,
PBGD, or CPPO in plants. In particular, the invention relates to plant genes that
encode a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity. The invention
has various utilities, including the recombinant production of polypeptides having
ENR-A, CBL, UROD, PBGD, or CPPO activity in heterologous hosts, the screening of chemicals
for herbicidal activity, and the use of thereby identified herbicidal chemicals to
control the growth of undesired vegetation. The invention may also be applied to the
development of herbicide tolerance in plants, plant tissues, plant seeds, and plant
cells.
[0002] The use of herbicides to control undesirable vegetation such as weeds in crop fields
has become almost a universal practice. The herbicide market exceeds 15 billion dollars
annually. Despite this extensive use, weed control remains a significant and costly
problem for farmers.
[0003] For example, present herbicides often impose special limitations on farming practices,
and the time and method of application and stage of weed plant development often are
critical for good weed control with such herbicides, thus creating farm management
constraints. Furthermore, since only a few target enzymes are inhibited by currently
used herbicides, various weed species are, or may become, resistant to these herbicides.
For all of these reasons, the discovery and development of effective new herbicides,
in particular those acting on novel target enzymes, is increasingly important.
[0004] Novel herbicides can now be discovered using high-throughput screens that implement
recombinant DNA technology. Once identified, metabolic enzymes essential to plant
growth and development can be recombinantly produced through standard molecular biological
techniques and utilized as herbicide targets in screens for novel inhibitors of the
enzyme's activity. The novel inhibitors discovered through such screens may then be
used as herbicides to control undesirable vegetation. Such herbicides are also useful
for selecting herbicide tolerant plants, and seed plants tolerant to the herbicide
can be produced, for example by genetic engineering techniques. Thus, herbicides that
exhibit greater potency, broader weed spectrum, and more rapid degradation in soil
can be applied to crops that are resistant or tolerant to herbicides in order to kill
weeds without attendant risk of damage to the crop.
[0005] Therefore, in order to meet the future food requirements of the world's growing population
in a cost-effective and environmentally safe manner, there exists a long felt and
unfulfilled need for novel target enzymes for herbicides, for new and better herbicides
inhibiting such target enzymes and for plants tolerant to these new and better herbicides.
[0006] The present invention thus provides:
isolated DNA molecules comprising
a nucleotide sequence encoding an amino acid sequence substantially similar to SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, but particularly to
SEQ ID NO:6 or SEQ ID NO:10
a nucleotide sequence substantially similar to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7 or SEQ ID NO:9, but particularly to SEQ ID NO:5, or SEQ ID NO:9
in particular
- wherein said nucleotide sequence is a plant nucleotide sequence
- wherein the amino acid sequence has ENR-A, CBL, UROD, PBGD, or CPPO activity, but
particularly UROD or CPPO activity
[0007] The present invention further provides a polypeptide comprising an amino acid sequence
encoded by a nucleotide sequence substantially similar to SEQ ID NO:1 , SEQ ID NO:3
, SEQ ID NO:5 , SEQ ID NO:7 or SEQ ID NO:9, in particular wherein
• the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence
substantially similar to SEQ ID NO:5 or SEQ ID NO:9
• said amino acid sequence is substantially similar to SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8 or SEQ ID NO:10
• said amino acid sequence is substantially similar to SEQ ID NO:6 or SEQ ID NO:10
• said amino acid sequence has ENR-A, CBL, UROD, PBGD, or CPPO activity, but particularly
UROD or CPPO activity
[0008] Further comprised by the present invention are polypeptides comprising an amino acid
sequence comprising at least 20, particularly 50 and more particularly 100 consecutive
amino acid residues of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8 or SEQ ID NO:10, but particularly of SEQ ID NO:6 or SEQ ID NO:10.
Further provided are
• expression cassettes comprising a promoter operatively linked to a DNA molecule
according to the invention and recombinant vectors comprising said expression cassettes
• host cells comprising a DNA molecule according to the invention, in particular,
wherein said host cell is selected from the group consisting of an insect cell, a
yeast cell
• prokaryotic cells and plant cells, plants or seeds comprising a plant cell according
to the invention
• plants according to the invention, wherein said plants are tolerant to an inhibitor
of ENR-A, CBL, UROD, PBGD, or CPPO activity, but particularly to an inhibitor of UROD
or CPPO activity
[0009] Further provided are
• expression cassettes comprising a promoter functional in a eukaryote operatively
linked to a DNA molecule comprising a nucleotide sequence substantially similar to
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9, but particularly
to SEQ ID NO:1 or SEQ ID NO:7
• recombinant vectors comprising said expression cassettes
• host cells comprising said expression cassette, in particular wherein said host
cells are selected from the group consisting of insect cells, yeast cells, prokaryotic
cells and plant cells
• plant cells comprising an isolated DNA molecule comprising a nucleotide sequence
identical or substantially similar to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7 or SEQ ID NO:9, but particularly to SEQ ID NO:1 or SEQ ID NO:7
• plants or seed comprising a plant cell of the invention
• plants according to the invention, wherein said plants are tolerant to an inhibitor
of ENR-A, CBL, UROD, PBGD, or CPPO activity, but particularly to an inhibitor of ENR-A
or PBGD activity
• host cells comprising an expression cassette, comprising a promoter operatively
linked to an isolated DNA molecule comprising a nucleotide sequence substantially
similar to SEQ ID NO:3, wherein said host cell is an eukaryotic cell
• a host cell as mentioned hereinbefore, wherein said host cell is selected from the
group consisting of an insect cell, a yeast cell, and a plant cell
• a plant or seed comprising a plant cell as mentioned hereinbefore
• a plant according to the invention, wherein said plant is tolerant to an inhibitor
of CBL activity
[0010] Further provided are methods comprising:
a) combining a polypeptide comprising the amino acid sequence encoded by a nucleotide
sequence substantially similar to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
or SEQ ID NO:9, or a homolog thereof, and a compound to be tested for the ability
to interact with said polypeptide, under conditions conducive to interaction; and
b) selecting a compound identified in step (a) that is capable of interacting with
said polypeptide and, optionally, further comprising:
c) applying a compound selected in step (b) to a plant to test for herbicidal activity;
and
d) selecting compounds having herbicidal activity.
[0011] Also provided are compounds identifiable by a method according to the invention,
in particular compounds having herbicidal activity.
[0012] Further provided is a process of identifying an inhibitor of ENR-A, CBL, UROD, PBGD,
or CPPO activity comprising:
a) introducing a DNA molecule comprising a nucleotide sequence substantially similar
to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, and encoding
a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity, or a homolog thereof,
into a plant cell, such that said sequence is functionally expressible at levels that
are higher than wild-type expression levels;
b) combining said plant cell with a compound to be tested for the ability to inhibit
the ENR-A, CBL, UROD, PBGD, or CPPO activity under conditions conducive to such inhibition;
c) measuring plant cell growth under the conditions of step (b);
d) comparing the growth of said plant cell with the growth of a plant cell having
unaltered ENR-A, CBL, UROD, PBGD, or CPPO activity under identical conditions; and
e) selecting said compound that inhibits plant cell growth in step (d) and compounds
having herbicidal activity identifiable according to the process of the invention.
[0013] In view of these long felt yet unfulfilled needs, one object of the invention is
to provide a method for identifying new or improved herbicides. Another object of
the invention is to provide a method for using such new or improved herbicides to
suppress the growth of plants such as weeds. Still another object of the invention
is to provide improved crop plants, and seed thereof, that are tolerant to such new
or improved herbicides.
[0014] In furtherance of these and other objects, the present invention provides DNA molecules
comprising a nucleotide sequence, preferably isolated from a plant, that encode a
polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity. The inventors are the
first to demonstrate that the ENR-A, CBL, UROD, PBGD, or CPPO genes are essential
for the growth of a plant, and therefore are good target enzymes for identifying new
herbicides.
[0015] According to one embodiment, the present invention provides a DNA molecule comprising
a nucleotide sequence isolated from a plant that encodes the polypeptide set forth
in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEO ID NO:8, or SEQ ID NO:10.
For example, the DNA molecule of the invention may comprise a nucleotide sequence
set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, respectively.
In another example, the DNA molecule of the invention comprises a nucleotide sequence
that is substantially similar to any one of the coding sequence set forth in SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, and that encodes a polypeptide
having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively. Although a nucleotide
sequence provided in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID
NO:9, is isolated from
Arabidopsis thaliana, using the information provided by the present invention, other nucleotide sequences
that encode a polypeptide having ENR-A, CBL, UROD, PBGD, or CPPO activity are obtained
from other sources, e.g. from other plants, using standard methods known in the art.
[0016] The present invention also provides a nucleotide sequence construct comprising a
promoter operatively linked to a DNA molecule of the invention. Further, the present
invention provides methods to stably transform such a nucleotide sequence construct
into a host cell, and host cells comprising such a nucleotide sequence construct,
wherein the host cell is capable of expressing the DNA molecule encoding a polypeptide
having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively. Any suitable cell may
be used as a host cell, e.g. a bacterial cell, a yeast cell, or a plant cell.
[0017] In accordance with another embodiment, the present invention also relates to the
recombinant production of a ENR-A, CBL, UROD, PBGD, or CPPO polypeptide and methods
of use of ENR-A, CBL, UROD, PBGD, or CPPO in assays for identifying compounds that
interact with ENR-A, CBL, UROD, PBGD, or CPPO polypeptide, respectively. In a preferred
embodiment, the present invention provides a plant polypeptide having ENR-A, CBL,
UROD, PBGD, or CPPO activity useful for identifying inhibitors of ENR-A, CBL, UROD,
PBGD, or CPPO activity, respectively, in
in vivo and
in vitro assays. Preferably the isolated polypeptide of the present invention comprises an
amino acid sequence substantially similar to any one of the amino acid sequence set
forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, respectively.
More preferably, this enzyme comprises the amino acid sequence set forth in SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
[0018] The present invention further provides methods of using purified polypeptides having
ENR-A, CBL, UROD, PBGD, or CPPO activity, preferably polypeptides derived from plant
sources, in assays to screen for and identify compounds that interact with a ENR-A,
CBL, UROD, PBGD, or CPPO polypeptide, respectively. Such compounds are preferably
inhibitors of ENR-A, CBL, UROD, PBGD, or CPPO activity, and are potentially herbicides
of future commercial interest. The inhibitors are used as herbicides to suppress the
growth of undesirable vegetation in fields where crops are grown, particularly agronomically
important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum,
rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar
cane, sugar beet, oilseed rape, and soybeans.
[0019] Thus, an assay useful for identifying inhibitors of essential plant genes, such as
plant ENR-A, CBL, UROD, PBGD, or CPPO genes, comprises the steps of:
a) reacting a plant ENR-A, CBL, UROD, PBGD, or CPPO enzyme, and a substrate thereof
in the presence of a suspected inhibitor of the enzyme's function;
b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor
to the rate of enzymatic activity under the same conditions in the absence of the
suspected inhibitor; and
c) determining whether the suspected inhibitor inhibits the ENR-A, CBL, UROD, PBGD,
or CPPO enzyme, respectively.
[0020] For example, the inhibitory effect on plant ENR-A, CBL, UROD, PBGD, or CPPO may be
determined by a reduction or complete inhibition of ENR-A, CBL, UROD, PBGD, or CPPO
activity in the assay. Such a determination may be made by comparing, in the presence
and absence of the candidate inhibitor, the amount of substrate used or intermediate
or product made during the reaction.
[0021] The present invention further embodies plants, plant tissues, plant seeds, and plant
cells that have modified ENR-A, CBL, UROD, PBGD, or CPPO activity, and that are therefore
tolerant to inhibition by a chemical at levels normally inhibitory to naturally occurring
ENR-A, CBL, UROD, PBGD, or CPPO enzyme activity, respectively. Herbicide tolerant
plants encompassed by the invention include those that would otherwise be potential
targets for normally inhibiting herbicides, particularly the agronomically important
crops mentioned above. According to one aspect of this embodiment, plants, plant tissue,
plant seeds, or plant cells are stably transformed with a recombinant DNA molecule
comprising a suitable promoter functional in plants operatively linked to a nucleotide
sequence that encodes an enzyme having modified ENR-A, CBL, UROD, PBGD, or CPPO activity,
that is tolerant to a concentration of a ENR-A, CBL, UROD, PBGD, or CPPO inhibitor,
respectively, that would normally inhibit the activity of wild-type, unmodified ENR-A,
CBL, UROD, PBGD, or CPPO, in the plant. Modified ENR-A, CBL, UROD, PBGD, or CPPO activity,
may also be conferred upon a plant by increasing expression of wild-type (i.e. sensitive)
ENR-A, CBL, UROD, PBGD, or CPPO enzyme, by providing multiple copies of wild-type
ENR-A, CBL, UROD, PBGD, or CPPO genes, to the plant or by overexpression of the endogenous
wild-type ENR-A, CBL, UROD, PBGD, or CPPO gene, or genes, under control of a stronger-than-wild-type
promoter, e.g., either a promoter that drives expression at a higher rate, or a promoter
that drives expression for a longer duration. The transgenic plants, plant tissue,
plant seeds, or plant cells thus created are then selected by conventional selection
techniques, whereby inhibitor tolerant descendants (lines) are isolated, characterized,
and developed. Alternately, random or site-specific mutagenesis may be used to generate
ENR-A, CBL, UROD, PBGD, or CPPO inhibitor tolerant lines. Still further, inhibitor
tolerant lines can be developed via selection of natural variants.
[0022] Therefore, the present invention provides a plant, plant cell, plant seed, or plant
tissue comprising a DNA molecule comprising a nucleotide sequence, preferably isolated
from a plant, that encodes an enzyme having ENR-A, CBL, UROD, PBGD, or CPPO activity,
and wherein the DNA molecule confers upon the plant, plant cell, plant seed, or plant
tissue tolerance to a ENR-A, CBL, UROD, PBGD, or CPPO inhibitor, in amounts that normally
naturally occurring ENR-A, CBL, UROD, PBGD, or CPPO activity. According to one example
of this embodiment, the enzyme comprises an amino acid sequence substantially similar
to any one of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, or SEQ ID NO:10. According to another example of this embodiment,
the DNA molecule is substantially similar to any one of the coding sequence set forth
in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. In a related
aspect, the present invention is directed to a method for selectively suppressing
the growth of weeds in a field containing a crop of planted crop seeds or plants,
comprising applying to crops or crop seeds that are tolerant to an inhibitor that
inhibits naturally occurring ENR-A, CBL, UROD, PBGD, or CPPO activity, and the weeds
in the field an ENR-A, CBL, UROD, PBGD, or CPPO inhibitor, respectively, in amounts
that inhibit naturally occurring ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively,
wherein the inhibitor suppresses the growth of the weeds without significantly suppressing
the growth of the crops.
[0023] Other objects and advantages of the present invention will become apparent to those
skilled in the art from a study of the following description of the invention and
non-limiting examples.
[0024] The invention thus provides:
An isolated DNA molecule comprising a nucleotide sequence substantially similar to
any one of SEQ ID NO:1, SEO ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. In
a preferred embodiment, the nucleotide sequence encodes an amino acid sequence substantially
similar to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID
NO:10. In another preferred embodiment, the nucleotide sequence is SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. In yet another preferred embodiment,
the nucleotide sequence encodes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4,
SEO ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. Preferably, the nucleotide sequence is
a plant nucleotide sequence, which preferably encodes a polypeptide having ENR-A,
CBL, UROD, PBGD, or CPPO activity.
[0025] The invention further provides:
A polypeptide comprising an amino acid sequence encoded by a nucleotide sequence substantially
similar to any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID
NO:9 . Preferably, the amino acid sequence is encoded by SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9. Preferably, the polypeptide comprises an
amino acid sequence substantially similar to any one of SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. Preferably the amino acid sequence is SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. The amino acid sequence
preferably has ENR-A, CBL, UROD, PBGD, or CPPO activity. In another preferred embodiment,
the amino acid sequence comprises at least 20 consecutive amino acid residues of the
amino acid sequence encoded by any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, or SEQ ID NO:9. Or, alternatively, the amino acid sequence comprises at least
20 consecutive amino acid residues of the amino acid sequence of any one of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
[0026] The invention further provides:
An expression cassette comprising a promoter operatively linked to a DNA molecule
according to the present invention, wherein the promoter is preferably functional
in a eukaryote, wherein the promoter is preferably heterologous to the DNA molecule.
The present invention further provides a recombinant vector comprising an expression
cassette according to the present invention, wherein said vector is preferably capable
of being stably transformed into a host cell, a host cell comprising a DNA molecule
according to the present invention, wherein said DNA molecule is preferably expressible
in the cell. The host cell is preferably selected from the group consisting of an
insect cell, a yeast cell, a prokaryotic cell and a plant cell. The invention further
provides a plant or seed comprising a plant cell of the present invention, wherein
the plant or seed is preferably tolerant to an inhibitor of ENR-A, CBL, UROD, PBGD,
or CPPO activity.
[0027] The invention further provides:
A process for making nucleotides sequences encoding gene products having altered ENR-A,
CBL, UROD, PBGD, or CPPO activity, comprising: a) shuffling an unmodified nucleotide
sequence of the present invention, b) expressing the resulting shuffled nucleotide
sequences, and c) selecting for altered ENR-A, CBL, UROD, PBGD, or CPPO activity,
as compared to the ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively, of the
gene product of said unmodified nucleotide sequence.
[0028] In a preferred embodiment, the unmodified nucleotide sequence is identical or substantially
similar to any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID
NO:9, or a homolog thereof. The present invention further provides a DNA molecule
comprising a shuffled nucleotide sequence obtainable by the process described above,
a DNA molecule comprising a shuffled nucleotide sequence produced by the process described
above. Preferably, a shuffled nucleotide sequence obtained by the process described
above has enhanced tolerance to an inhibitor of ENR-A, CBL, UROD, PBGD, or CPPO activity.
The invention further provides an expression cassette comprising a promoter operatively
linked to a DNA molecule comprising a shuffled nucleotide sequence a recombinant vector
comprising such an expression cassette, wherein said vector is preferably capable
of being stably transformed into a host cell, a host cell comprising such an expression
cassette, wherein said nucleotide sequence is preferably expressible in said cell.
A preferred host cell is selected from the group consisting of an insect cell, a yeast
cell, a prokaryotic cell and a plant cell. The invention further provides a plant
or seed comprising such plant cell, wherein the plant is preferably tolerant to an
inhibitor of ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively.
[0029] The invention further provides:
A method for selecting compounds that interact with the protein encoded by SEO ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, comprising: a) expressing
a DNA molecule comprising SEQ ID NO:1, SEO ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ
ID NO:9, or a sequence substantially similar to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, or SEO ID NO:9, or a homolog thereof, to generate the corresponding protein,
b) testing a compound suspected of having the ability to interact with the protein
expressed in step (a), and c) selecting compounds that interact with the protein in
step (b).
[0030] The invention further provides:
A process of identifying an inhibitor of ENR-A, CBL, UROD, PBGD, or CPPO activity,
comprising: a) introducing a DNA molecule comprising a nucleotide sequence of SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, and having ENR-A,
CBL, UROD, PBGD, or CPPO activity, or nucleotide sequences substantially similar thereto,
or a homolog thereof, into a plant cell, such that said sequence is functionally expressible
at levels that are higher than wild-type expression levels, b) combining said plant
cell with a compound to be tested for the ability to inhibit the ENR-A, CBL, UROD,
PBGD, or CPPO activity, respectively, under conditions conducive to such inhibition,
c) measuring plant cell growth under the conditions of step (b),
d) comparing the growth of said plant cell with the growth of a plant cell having
unaltered ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively, under identical
conditions, and
e) selecting said compound that inhibits plant cell growth in step (d).
[0031] The invention further comprises a compound having herbicidal activity identifiable
according to the process described immediately above.
[0032] The invention further comprises:
A process of identifying compounds having herbicidal activity comprising:
a) combining a protein of the present invention and a compound to be tested for the
ability to interact with said protein, under conditions conducive to interaction,
b) selecting a compound identified in step (a) that is capable of interacting with
said protein, c) applying identified compound in step (b) to a plant to test for herbicidal
activity, and d) selecting compounds having herbicidal activity.
[0033] The invention further comprises a compound having herbicidal activity identifiable
according to the process described immediately above.
[0034] The invention further comprises:
A method for suppressing the growth of a plant comprising, applying to said plant
a compound that inhibits the activity of a polypeptide of the present invention in
an amount sufficient to suppress the growth of said plant.
[0035] The invention further comprises:
A method for recombinantly expressing a protein having ENR-A, CBL, UROD, PBGD, or
CPPO activity comprising introducing a nucleotide sequence encoding a protein having
one of the above activities into a host cell and expressing the nucleotide sequence
in the host cell. A preferred host cell is selected from the group consisting of an
insect cell, a yeast cell, a prokaryotic cell and a plant cell. A preferred prokaryotic
cell is a bacterial cell, e.g.
E. coli.
BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
[0036]
SEQ ID NO:1 is a cDNA sequence encoding ENR-A from Arabidopsis thaliana.
SEQ ID NO:2 is the predicted amino acid sequence of Arabidopsis thaliana ENR-A encoded by SEQ ID NO:1.
SEQ ID NO:3 is cDNA coding sequence for the CBL gene from Arabidopsis thaliana.
SEQ ID NO:4 amino acid sequence encoded by the Arabidopsis thaliana CBL sequence shown in SEQ ID NO:3.
SEQ ID NO:5 is a cDNA sequence encoding UROD from Arabidopsis thaliana.
SEQ ID NO:6 is the predicted amino acid sequence of Arabidopsis thaliana UROD encoded by SEQ ID NO:5.
SEQ ID NO:7 is a cDNA sequence encoding PBGD from Arabidopsis thaliana.
SEQ ID NO:8 is the predicted amino acid sequence of Arabidopsis thaliana PBGD encoded by SEQ ID NO:7.
SEQ ID NO:9 is a cDNA sequence encoding CPPO from Arabidopsis thaliana.
SEQ ID NO:10 is the predicted amino acid sequence of Arabidopsis thaliana CPPO encoded by SEQ ID NO:9.
SEQ ID NO:11 is the genomic sequence of the ENR-A gene from Arabidopsis thaliana.
SEQ ID NO:12 is the oligonucleotide ENR-A-F2
SEQ ID NO:13 is the oligonucleotide ENR-A-R2
SEQ ID NO:14 is the sequence for oligonucleotide DG354.
SEQ ID NO:15 is the sequence for oligonucleotide DG357.
SEQ ID NO:16 is the sequence for oligonucleotide CBL1
SEQ ID NO:17 is the sequence for oligonucleotide CBL2.
SEQ ID NO:18 is the sequence for oligonucleotide CBL3.
SEQ ID NO:19 is the sequence for oligonucleotide ASV1.
SEQ ID NO:20 is the sequence for oligonucleotide ASV2.
SEQ ID NO:21 is the genomic sequence of Arabidopsis thaliana UROD
SEQ ID NO:22 is the sequence for oligonucleotide UROD-N-Nde
SEQ ID NO:23 is the sequence for oligonucleotide UROD-C-Not
SEQ ID NO:24 is the sequence for oligonucleotide UROD-F2
SEQ ID NO:25 is the sequence for oligonucleotide UROD-R2
SEQ ID NO:26 is the genomic sequence of Arabidopsis thaliana PBGD.
SEQ ID NO:27 is the sequence for oligonucleotide PORD-F2.
SEQ ID NO:28 is the sequence for oligonucleotide PORD-R2.
SEQ ID NO:29 is the genomic sequence of the CPPO gene from Arabidopsis thaliana.
SEQ ID NO:30 is the sequence for oligonucleotide CR73.
SEQ ID NO:31 is the sequence for oligonucleotide CR75.
SEQ ID NO:32 is the sequence for oligonucleotide JG-L.
SEQ ID NO:33 is the sequence for oligonucleotide CPPGO-F2.
SEQ ID NO:34 is the sequence for oligonucleotide CPPGO-R2.
[0037] For clarity, certain terms used in the specification are defined and used as follows:
Activatable DNA Sequence: a DNA sequence that regulates the expression of genes in a genome, desirably the
genome of a plant. The activatable DNA sequence is complementary to a target gene
endogenous in the genome, in this case the gene encoding ENR-A, CBL, UROD, PBGD, or
CPPO. When the activatable DNA sequence is introduced and expressed in a cell, it
inhibits expression of the target gene. An activatable DNA sequence useful in conjunction
with the present invention includes those encoding or acting as dominant inhibitors,
such as a translatable or untranslatable sense sequence capable of disrupting gene
function in stably transformed plants to positively identify one or more genes essential
for normal growth and development of a plant. A preferred activatable DNA sequence
is an antisense DNA sequence. The interaction of the antisense sequence and the target
gene results in substantial inhibition of the expression of the target gene so as
to kill the plant, or at least inhibit normal plant growth or development.
Activatable DNA Construct: a recombinant DNA construct comprising a synthetic promoter operatively linked to
the activatable DNA sequence, which when introduced into a cell, desirably a plant
cell, is not expressed, i.e. is silent, unless a complete hybrid transcription factor
capable of binding to and activating the synthetic promoter is present. The activatable
DNA construct is introduced into cells, tissues, or plants to form stable transgenic
lines capable of expressing the activatable DNA sequence.
Antiparallel: "Antiparallel" refers herein to two nucleotide sequences paired through hydrogen
bonds between complementary base residues with phosphodiester bonds running in the
5'-3' direction in one nucleotide sequence and in the 3'-5' direction in the other
nucleotide sequence.
Co-factor: natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed
reaction. A co-factor is e.g. NAD(P), riboflavin (including FAD and FMN), folate,
molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine,
pyridoxal phosphate, ubiquinone, menaquinone. Optionally, a co-factor can be regenerated
and reused.
Complementary: "Complementary" refers to two nucleotide sequences which comprise antiparallel nucleotide
sequences capable of pairing with one another upon formation of hydrogen bonds between
the complementary base residues in the antiparallel nucleotide sequences.
DNA shuffling: DNA shuffling is a method to rapidly, easily and efficiently introduce mutations
or rearrangements, preferably randomly, in a DNA molecule or to generate exchanges
of DNA sequences between two or more DNA molecules, preferably randomly. The DNA molecule
resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring
DNA molecule derived from at least one template DNA molecule. The shuffled DNA encodes
an enzyme modified with respect to the enzyme encoded by the template DNA, and preferably
has an altered biological activity with respect to the enzyme encoded by the template
DNA.
Enzyme activity: means herein the ability of an enzyme to catalyze the conversion of a substrate
into a product. A substrate for the enzyme comprises the natural substrate of the
enzyme but also comprises analogues of the natural substrate, which can also be converted,
by the enzyme into a product or into an analogue of a product. The activity of the
enzyme is measured for example by determining the amount of product in the reaction
after a certain period of time, or by determining the amount of substrate remaining
in the reaction mixture after a certain period of time. The activity of the enzyme
is also measured by determining the amount of an unused co-factor of the reaction
remaining in the reaction mixture after a certain period of time or by determining
the amount of used co-factor in the reaction mixture after a certain period of time.
The activity of the enzyme is also measured by determining the amount of a donor of
free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate
or phosphocreatine) remaining in the reaction mixture after a certain period of time
or by determining the amount of a used donor of free energy or energy-rich molecule
(e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture after a certain
period of time.
Essential: An "essential" gene is a gene encoding a protein such as e.g. a biosynthetic enzyme,
receptor, signal transduction protein, structural gene product, or transport protein
that is essential to the growth or survival of the plant.
Expression cassette: "Expression cassette" as used herein means a DNA sequence capable of directing expression
of a particular nucleotide sequence in an appropriate host cell, comprising a promoter
operably linked to the nucleotide sequence of interest which is operably linked to
termination signals. It also typically comprises sequences required for proper translation
of the nucleotide sequence. The coding region usually codes for a protein of interest
but may also code for a functional RNA of interest, for example antisense RNA or a
nontranslated RNA, in the sense or antisense direction. The expression cassette comprising
the nucleotide sequence of interest may be chimeric, meaning that at least one of
its components is heterologous with respect to at least one of its other components.
The expression cassette may also be one which is naturally occurring but has been
obtained in a recombinant form useful for heterologous expression. Typically, however,
the expression cassette is heterologous with respect to the host, i.e., the particular
DNA sequence of the expression cassette does not occur naturally in the host cell
and must have been introduced into the host cell or an ancestor of the host cell by
a transformation event. The expression of the nucleotide sequence in the expression
cassette may be under the control of a constitutive promoter or of an inducible promoter
which initiates transcription only when the host cell is exposed to some particular
external stimulus. In the case of a multicellular organism, such as a plant, the promoter
can also be specific to a particular tissue or organ or stage of development. In the
case of a plastid expression cassette, for expression of the nucleotide sequence from
a plastid genome, additional elements, i.e. ribosome binding sites, may be required.
Herbicide: a chemical substance used to kill or suppress the growth of plants, plant cells,
plant seeds, or plant tissues.
Heterologous DNA Sequence: a DNA sequence not naturally associated with a host cell into which it is introduced,
including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
Homologous DNA Sequence: a DNA sequence naturally associated with a host cell.
Inhibitor: a chemical substance that inactivates the enzymatic activity of ENR-A, CBL, UROD,
PBGD, or CPPO. The term "herbicide" is used herein to define an inhibitor when applied
to plants, plant cells, plant seeds, or plant tissues.
Isogenic: plants which are genetically identical, except that they may differ by the presence
or absence of a heterologous DNA sequence.
Isolated: in the context of the present invention, an isolated DNA molecule or an isolated
enzyme is a DNA molecule or enzyme which, by the hand of man, exists apart from its
native environment and is therefore not a product of nature. An isolated DNA molecule
or enzyme may exist in a purified form or may exist in a non-native environment such
as, for example, in a transgenic host cell.
Mature protein: protein which is normally targeted to a cellular organelle, such as a chloroplast,
and from which the transit peptide has been removed.
Minimal Promoter: promoter elements, particularly a TATA element, that are inactive or that have greatly
reduced promoter activity in the absence of upstream activation. In the presence of
a suitable transcription factor, the minimal promoter functions to permit transcription.
Modified Enzyme Activity: enzyme activity different from that which naturally occurs in a plant (i.e. enzyme
activity that occurs naturally in the absence of direct or indirect manipulation of
such activity by man), which is tolerant to inhibitors that inhibit the naturally
occurring enzyme activity.
Native: A "native" refers to a gene which is present in the genome of the untransformed
plant cell.
Plant: A "plant" refers to any plant or part of a plant at any stage of development. Therein
are also included cuttings, cell or tissue cultures and seeds. As used in conjunction
with the present invention, the term "plant tissue" includes, but is not limited to,
whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures,
and any groups of plant cells organized into structural and/or functional units.
Significant Increase: an increase in enzymatic activity that is larger than the margin of error inherent
in the measurement technique, preferably an increase by about 2-fold or greater of
the activity of the wild-type enzyme in the presence of the inhibitor, more preferably
an increase by about 5-fold or greater, and most preferably an increase by about 10-fold
or greater.
With respect to CBL, in its broadest sense, the term "substantially similar", when
used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding
to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the polypeptide encoded by
the reference nucleotide sequence, e.g. where only changes in amino acids not affecting
the polypeptide function occur. Desirably the substantially similar nucleotide sequence
encodes the polypeptide encoded by the reference nucleotide sequence. The term "substantially
similar" is specifically intended to include nucleotide sequences wherein the sequence
has been modified to optimize expression in particular cells. The percentage of identity
between the substantially similar nucleotide sequence and the reference nucleotide
sequence desirably is at least 65%, more desirably at least 75%, preferably at least
85%, more preferably at least 90%, still more preferably at least 95%, yet still more
preferably at least 99%. Sequence comparisons are carried out using a Smith-Waterman
sequence alignment algorithm (see e.g. Waterman, M.S. Introduction to Computational
Biology: Maps, sequences and genomes. Chapman & Hall. London: 1995. ISBN 0-412-99391-0,
or at
http://www-hto.usc.edu/software/seqaln/index.html). The locals program, version 1.16, is used with following parameters: match: 1,
mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. A nucleotide
sequence "substantially similar" to reference nucleotide sequence hybridizes to the
reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. As used herein the
term "CBL gene" refers to a DNA molecule comprising SEQ ID NO:3 or comprising a nucleotide
sequence substantially similar to SEO ID NO:3. Homologs of the CBL gene include nucleotide
sequences that encode an amino acid sequence that is at least 30% identical to SEQ
ID NO:4 as measured, using the parameters described below, wherein the amino acid
sequence encoded by the homolog has the biological activity of the CBL protein.
[0038] With respect to CBL, the term "substantially similar", when used herein with respect
to a protein, means a protein corresponding to a reference protein, wherein the protein
has substantially the same structure and function as the reference protein, e.g. where
only changes in amino acids sequence not affecting the polypeptide function occur.
When used for a protein or an amino acid sequence the percentage of identity between
the substantially similar and the reference protein or amino acid sequence desirably
is at least 65%, more desirably at least 75%, preferably at least 85%, more preferably
at least 90%, still more preferably at least 95%, yet still more preferably at least
99%, using default BLAST analysis parameters. As used herein the term "CBL protein"
refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide
sequence substantially similar to SEQ ID NO:3. Homologs of the CBL protein are amino
acid sequences that are at least 30% identical to SEQ ID NO:4, as measured using the
parameters described above, wherein the amino acid sequence encoded by the homolog
has the biological activity of the CBL protein.
[0039] With respect to UROD, in its broadest sense, the term "substantially similar", when
used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding
to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the polypeptide encoded by
the reference nucleotide sequence Desirably the substantially similar nucleotide sequence
encodes the polypeptide encoded by the reference nucleotide sequence. The term "substantially
similar" is specifically intended to include nucleotide sequences wherein the sequence
has been modified to optimize expression in particular cells. Preferably, "substantially
similar" refers to nucleotide sequences that encode a protein having at least 85%
identity to SEQ ID NO:6, wherein said protein sequence comparisons are conducted using
GAP analysis as described below. A nucleotide sequence "substantially similar" to
the reference nucleotide sequence hybridizes to the reference nucleotide sequence
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1 % SDS at 50°C, more preferably
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. As used herein the
term "UROD gene" refers to a DNA molecule comprising SEQ ID NO:5 or comprising a nucleotide
sequence substantially similar to SEQ ID NO:5. Homologs of the UROD gene include nucleotide
sequences that encode an amino acid sequence that is at least 30% identical to SEQ
ID NO:6 as measured, using the parameters described below, wherein the amino acid
sequence encoded by the homolog has the biological activity of the UROD protein. Preferable
are dicot homologs.
[0040] With respect to UROD, the term "substantially similar", when used herein with respect
to a protein, means a protein corresponding to a reference protein, wherein the protein
has substantially the same structure and function as the reference protein, e.g. where
only changes in amino acids sequence not affecting the polypeptide function occur.
When used for a protein or an amino acid sequence the percentage of identity between
the substantially similar and the reference protein or amino acid sequence desirably
is preferably at least 85%, more preferably at least 90%, still more preferably at
least 95%, yet still more preferably at least 99%, using default GAP analysis parameters
with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm
of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453). As
used herein the term "UROD protein" refers to an amino acid sequence encoded by a
DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:5.
Homologs of the UROD protein are amino acid sequences that are at least 30% identical
to SEQ ID NO:6, as measured using the parameters described above, wherein the amino
acid sequence encoded by the homolog has the biological activity of the UROD protein.
Preferable are dicot homologs.
[0041] With respect to PBGD, in its broadest sense, the term "substantially similar", when
used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding
to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the polypeptide encoded by
the reference nucleotide sequence. Desirably the substantially similar nucleotide
sequence encodes the polypeptide encoded by the reference nucleotide sequence. The
term "substantially similar" is specifically intended to include nucleotide sequences
wherein the sequence has been modified to optimize expression in particular cells.
Preferably, "substantially similar" refers to nucleotide sequences that encode a protein
having at least 85% identity to SEQ ID NO:8, wherein said protein sequence comparisons
are conducted using GAP analysis as described below. A nucleotide sequence "substantially
similar" to the reference nucleotide sequence hybridizes to the reference nucleotide
sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1 % SDS at 50°C, more desirably still
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. As used herein the
term "PBGD gene" refers to a DNA molecule comprising SEQ ID NO:7 or comprising a nucleotide
sequence substantially similar to SEQ ID NO:7. Homologs of the PBGD gene include nucleotide
sequences that encode an amino acid sequence that is at least 30% identical to SEQ
ID NO:8 as measured, using the parameters described below, wherein the amino acid
sequence encoded by the homolog has the biological activity of the PBGD protein. Preferable
are dicot homologs.
[0042] With respect to PBGD, the term "substantially similar", when used herein with respect
to a protein, means a protein corresponding to a reference protein, wherein the protein
has substantially the same structure and function as the reference protein, e.g. where
only changes in amino acids sequence not affecting the polypeptide function occur.
When used for a protein or an amino acid sequence the percentage of identity between
the substantially similar and the reference protein or amino acid sequence desirably
is preferably at least 85%, more preferably at least 90%, still more preferably at
least 95%, yet still more preferably at least 99%, using default GAP analysis parameters
with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm
of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453). As
used herein the term "PBGD protein" refers to an amino acid sequence encoded by a
DNA molecule comprising a nucleotide sequence substantially similar to SEQ ID NO:7.
Homologs of the PBGD protein are amino acid sequences that are at least 30% identical
to SEQ ID NO:8, as measured using the parameters described above, wherein the amino
acid sequence encoded by the homolog has the biological activity of the PBGD protein.
Preferable are dicot homologs.
[0043] With respect to CPPO, in its broadest sense, the term "substantially similar", when
used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding
to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the polypeptide encoded by
the reference nucleotide sequence. Desirably, the substantially similar nucleotide
sequence encodes the polypeptide encoded by the reference nucleotide sequence. The
term "substantially similar" is specifically intended to include nucleotide sequences
wherein the sequence has been modified to optimize expression in particular cells.
Preferably, "substantially similar" refers to nucleotide sequences that encode a protein
having at least 81% identity, more preferably at least 85% identity, still more preferably
at least 90% identity, still more preferably at least 95% identity, yet still more
preferably at least 99% identity, to SEQ ID NO:10, wherein said protein sequence comparisons
are conducted using GAP analysis as described below. Also, "substantially similar"
preferably also refers to nucleotide sequences having at least 75% identity, more
preferably at least 80% identity, still more preferably at least 85% identity, still
more preferably at least 90% identity, still more preferably 95% identity, yet still
more preferably at least 99% identity, to SEQ ID NO:9, wherein said nucleotide sequence
comparisons are conducted using GAP analysis as described below. A nucleotide sequence
"substantially similar" to the reference nucleotide sequence preferably hybridizes
to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. As used herein the
term "CPPO gene" refers to a DNA molecule comprising SEQ ID NO:9 or comprising a nucleotide
sequence substantially similar to SEQ ID NO:9. Homologs of the CPPO gene include nucleotide
sequences that encode an amino acid sequence that is at least 50% identical to SEQ
ID NO:10, more preferably at least 60% identical, still more preferably at least 65%
identical, still more preferably at least 70%, yet still more preferably at least
80%, as measured, using the parameters described below, wherein the amino acid sequence
encoded by the homolog has the biological activity of the CPPO protein.
[0044] With respect to CPPO, the term "substantially similar", when used herein with respect
to a protein, means a protein corresponding to a reference protein, wherein the protein
has substantially the same structure and function as the reference protein, e.g. where
only changes in amino acids sequence not affecting the polypeptide function occur.
When used for a protein or an amino acid sequence the percentage of identity between
the substantially similar and the reference protein or amino acid sequence desirably
is preferably at least 81%, more preferably at least 85%, still more preferably at
least 90%, more preferably at least 95%, still more preferably at least 99% using
default GAP analysis parameters with the University of Wisconsin GCG (version 10),
SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman
and Wunsch (1970) J Mol. Biol. 48: 443-453). As used herein the term "CPPO protein"
refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide
sequence substantially similar to SEQ ID NO:9. Homologs of the CPPO protein are amino
acid sequences that are at least 50% identical to SEQ ID NO:10, as measured using
the parameters described above, wherein the amino acid sequence encoded by the homolog
has the biological activity of the CPPO protein.
[0045] With respect to ENR-A, in its broadest sense, the term "substantially similar", when
used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding
to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide
having substantially the same structure and function as the polypeptide encoded by
the reference nucleotide sequence. Desirably, the substantially similar nucleotide
sequence encodes the polypeptide encoded by the reference nucleotide sequence. The
term "substantially similar" is specifically intended to include nucleotide sequences
wherein the sequence has been modified to optimize expression in particular cells.
Preferably, "substantially similar" refers to nucleotide sequences that encode a protein
having at least 90% identity, more preferably at least 95% identity, yet still more
preferably at least 99% identity, to SEQ ID NO:2, wherein said protein sequence comparisons
are conducted using GAP analysis as described below. Also, "substantially similar"
preferably also refers to nucleotide sequences having at least 85% identity, more
preferably at least 90% identity, still more preferably 95% identity, yet still more
preferably at least 99% identity, to SEQ ID NO:1, wherein said nucleotide sequence
comparisons are conducted using GAP analysis as described below. A nucleotide sequence
"substantially similar" to the reference nucleotide sequence preferably hybridizes
to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still
in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO
4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. As used herein the
term "ENR-A gene" refers to a DNA molecule comprising SEQ ID NO:1 or comprising a
nucleotide sequence substantially similar to SEO ID NO:1. Homologs of the ENR-A gene
include nucleotide sequences that encode an amino acid sequence that is at least 30%
identical to SEQ ID NO:2, more preferably at least 70%, still more preferably at least
85%, yet still more preferably at least 90%, as measured, using the parameters described
below, wherein the amino acid sequence encoded by the homolog has the biological activity
of the ENR-A protein.
[0046] With respect to ENR-A, the term "substantially similar", when used herein with respect
to a protein, means a protein corresponding to a reference protein, wherein the protein
has substantially the same structure and function as the reference protein, e.g. where
only changes in amino acids sequence not affecting the polypeptide function occur.
When used for a protein or an amino acid sequence the percentage of identity between
the substantially similar and the reference protein or amino acid sequence desirably
is preferably at least 90%, more preferably at least 95%, still more preferably at
least 99% using default GAP analysis parameters with the University of Wisconsin GCG
(version 10), SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch
(Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453). As used herein the term "ENR-A
protein" refers to an amino acid sequence encoded by a DNA molecule comprising a nucleotide
sequence substantially similar to SEQ ID NO:1. Homologs of the ENR-A protein are amino
acid sequences that are at least 30% identical to SEQ ID NO:2, as measured using the
parameters described above, wherein the amino acid sequence encoded by the homolog
has the biological activity of the ENR-A protein.
[0047] Substrate: a substrate is the molecule that an enzyme naturally recognizes and converts to
a product in the biochemical pathway in which the enzyme naturally carries out its
function, or is a modified version of the molecule, which is also recognized by the
enzyme and is converted by the enzyme to a product in an enzymatic reaction similar
to the naturally-occurring reaction.
[0048] Target gene: A "target gene" is any gene in a plant cell. For example, a target gene is a gene
of known function or is a gene whose function is unknown, but whose total or partial
nucleotide sequence is known. Alternatively, the function of a target gene and its
nucleotide sequence are both unknown. A target gene is a native gene of the plant
cell or is a heterologous gene which had previously been introduced into the plant
cell or a parent cell of said plant cell, for example by genetic transformation. A
heterologous target gene is stably integrated in the genome of the plant cell or is
present in the plant cell as an extrachromosomal molecule, e.g. as an autonomously
replicating extrachromosomal molecule.
[0049] Tolerance: the ability to continue essentially normal growth or function (i.e. no more than
5% of herbicide tolerant plants show phytotoxicity) when exposed to an inhibitor or
herbicide in an amount sufficient to suppress the normal growth or function of native,
unmodified plants.
[0050] Transformation: a process for introducing heterologous DNA into a cell, tissue, or plant. Transformed
cells, tissues, or plants are understood to encompass not only the end product of
a transformation process, but also transgenic progeny thereof.
[0051] Transgenic: stably transformed with a recombinant DNA molecule that preferably comprises a suitable
promoter operatively linked to a DNA sequence of interest.
I. Plant ENR-A, CBL, UROD, PBGD, or CPPO Genes, respectively
[0052] In the present invention, the following abbreviations are used for the above plant
genes. ENR-A is the abbreviation for enoyl-acyl carrier protein reductase; CBL is
the abbreviation for cystathionine beta lyase; UROD is the abbreviation for uroporphyrinogen
decarboxylase; PBGD is the abbreviation for porphobilinogen deaminase; and CPPO is
the abbreviation for coproporphyrinogen oxidase.
[0053] CBL (EC 4.4.1.8) is an enzyme catalyzing a biochemical reaction required for the
biosynthesis of the amino acid methionine. The methionine biosynthetic pathway in
plants is outlined in Figure 1 of Ravenel et al., (1998)
Proc. Natl. Acad. Sci. USA 95:7805-7812, incorporated herein by reference. This enzyme catalyzes the conversion
of cystathionine to homocysteine by cleaving cystathionine to produce homocysteine,
pyruvate, and ammonia. The sequence of a cDNA for the
Arabidopsis CBL gene has been identified (EMBL accession # L40511; Ravanel et al. (1995)
Plant Mol. Biol. 29: 875-882). The CBL gene has been cloned from other organisms, including
E. coli (SWISS PROT accession # P06721),
S. typhimurium (PIR accession # JV0020),
S. cerevisiae (SWISS PROT accession # P43623),
B. subtilis (GenPept accession # Z99110 AL009126),
Emericella nidulans (GenPept accession # U28383),and human (GenPept accession # S52784). Results from
GAP analysis of the above sequences show the following identities relative to
Arabidopsis thaliana:
E. coli (28% identical);
S. cerevisiae (28% identical); humans (41% identical);
B. subtilis (46% identical), and
Emericella nidulans (47% identical).
[0054] UROD (EC 4.1.1.37) is an enzyme catalyzing a biochemical reaction required for the
biosynthesis of porphyrin and heme. The porphyrin biosynthetic pathway in plants is
outlined in Figure 1 of Reinbothe & Reinbothe,
Plant Physiol. 111:1-7 (1996), incorporated herein by reference. This enzyme catalyzes the conversion
of uroporphyrinogen III to coproporphyrinogen III. Coproporphyrinogen III is synthesized
by plants, microorganisms, and animals as a precursor for the production of porphyrin
and heme. In most organisms, heme is required as a prosthetic group for many enzymes,
e.g. cytochrome oxidase. In plants, the porphyrin pathway produces chlorophyll (reviewed
in Suzuki et al.,
Annu. Rev.
[0055] Genet. 31:61-89 (1997) and Reinbothe & Reinbothe,
Plant Physiol. 111:1-7 (1996). The UROD gene has been cloned from many organisms, including
E. coli (SWISS PROT accession # P29680),
S. cerevisiae (SWISS PROT accession # P32347), humans (SWISS PROT accession # P06132), maize (SWISS
PROT accession # 081220), and tobacco (SWISS PROT accession # Q42967).
[0056] PBGD (also known as hydroxymethylbilane synthase or preuroporphyrinogen synthase)
(EC 4.3.1.8) is an enzyme catalyzing a biochemical reaction required for the biosynthesis
of porphyrin and heme. The porphyrin biosynthetic pathway in plants is outlined in
Figure 1 of Reinbothe & Reinbothe,
Plant Physiol. 111:1-7 (1996), incorporated herein by reference. This enzyme catalyzes the condensation
of four molecules of porphobilinogen to form hydroxymethylbilane. Hydroxymethylbilane
is synthesized by plants, microorganisms, and animals as a precursor for the production
of porphyrin and heme. In most organisms, heme is required as a prosthetic group for
many enzymes, e.g. cytochrome oxidase. In plants, the porphyrin pathway produces chlorophyll
(reviewed in Suzuki et al.,
Annu. Rev. Genet. 31:61-89 (1997) and Reinbothe & Reinbothe,
Plant Physiol. 111:1-7 (1996). The PBGD gene has been cloned from many organisms, including
E. coli (SWISS PROT accession # P06983),
S. cerevisiae (SWISS PROT accession # P28789), humans (SWISS PROT accession # P08397), pea (SWISS
PROT accession #Q43082),
Methanococcus jannaschii (SWISS PROT accession #Q57989), and
Arabidopsis thaliana (SWISS PROT accession # Q43316) (Lim et al.,
Plant Mol. Biol. 26:863-872 (1994)). Results from GAP analysis of the above sequences show the following
identities relative to
Arabidopsis thaliana:
E. coli (45% identical);
S. cerevisiae (35% identical); humans (37% identical); pea (78% identical), and
Methanococcus jannaschii (41% identical).
[0057] CPPO (EC 1.3.3.3) is an enzyme catalyzing a biochemical reaction required for the
biosynthesis of porphyrin and heme. The porphyrin biosynthetic pathway in plants is
outlined in Figure 1 of Reinbothe & Reinbothe,
Plant Physiol. 111:1-7 (1996), incorporated herein by reference. This enzyme catalyzes the conversion
of coproporphyrinogen III to protoporphyrinogen IX. Protoporphyrinogen IX is synthesized
by plants, microorganisms, and animals as a precursor for the production of porphyrin
and heme. In most organisms, heme is required as a prosthetic group for many enzymes,
e.g. cytochrome oxidase. In plants, the porphyrin pathway produces chlorophyll (reviewed
in Suzuki et al.,
Annu. Rev. Genet. 31:61-89 (1997) and Reinbothe & Reinbothe,
Plant Physiol. 111:1-7 (1996). The CPPO gene has been cloned from many organisms, including
E. coli, aerobic form (SWISS PROT accession #P36553),
S. cerevisiae (SWISS PROT accession # P11353), humans (SWISS PROT accession # P36551), barley (SWISS
PROT accession #Q42480), tobacco (SWISS PROT accession # Q42946), and soybean (SWISS
PROT accession #P35055). Results from GAP analysis of the above sequences show the
following identities at the amino acid level relative to
Arabidopsis thaliana: E. coli, aerobic form (48% identical),
S. cerevisiae (52% identical), humans (53% identical), barley (75% identical), tobacco (79% identical),
and soybean (80% identical), and the following identities at the nucleotide level
relative to
Arabidopsis thaliana: barley (65% identical), soybean (73% identical).
[0058] ENR-A, also known as NADH enoyl-ACP reductase, (EC 1.3.1.9) is an enzyme catalyzing
a biochemical reaction required for the final reducing step in the fatty acid biosynthesis
cycle. The fatty acid biosynthetic pathway in plants is outlined in Figure 6.6 of
Dey & Harborne,
Plant Biochemistry, Academic Press (1997) incorporated herein by reference. This enzyme catalyzes the reduction of enoyl-acyl-ACP
derivatives of carbon chain length from 4 to 16, by reducing a
trans-unsaturated double bond to produce a saturated acyl-ACP, which can be elongated in
the next condensation reaction. In plants, fatty acids act as energy stores, membrane
constituents, and play key roles in metabolic control via second messenger signaling.
The ENR-A gene has been cloned from many organisms, including
E. coli (SWISS PROT accession #P29132), Petunia (GenBank accession # CAA05879), rice (GenBank
accession # CAA05816),
Arabidopsis thaliana (GenBank accession # CAA74175), and rape (SWISS PROT accession #P80030). Results
from GAP analysis of the above sequences show the following identities at the amino
acid level relative to
Arabidopsis thaliana: E. coli (34% identical), Petunia (71% identical), rice (73% identical), and rape (90% identical),
and the following identities at the nucleotide level relative to
Arabidopsis thaliana: rape (85% identical). The sequences controlling the expression of the
Arabidopsis thaliana enr-A gene have been described (de Boer, G.-J. et. al. (1999) Plant Mol. Biol. 39:1197-1207).
The corresponding
E. coli gene,
Fabl, has been shown to be inhibited by triclosan, an antimicrobial biocide (Heath, R.J.
et al. (1999) J. Biol. Chem. 274:11110-11114).
[0059] In one aspect, the present invention is directed to a DNA molecule comprising a nucleotide
sequence isolated from a plant source that encodes ENR-A, CBL, UROD, PBGD, or CPPO.
In particular, the present invention provides a DNA molecule isolated from
Arabidopsis thaliana that encodes ENR-A, CBL, UROD, PBGD, or CPPO, and DNA molecules substantially similar
thereto that encode enzymes having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively.
The DNA coding sequence for ENR-A, CBL, UROD, PBGD, or CPPO, from
Arabidopsis thaliana is provided in SEQ ID NO:1, SEO ID NO:3, SEO ID NO:5, SEQ ID NO:7, or SEQ ID NO:9,
respectively. The DNA sequence of the genomic sequence of the UROD, PBGD, CPPO, or
ENR-A gene, from
Arabidopsis thaliana is set forth in SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:11, respectively.
[0060] Based on Applicants' disclosure of the present invention, ENR-A, CBL, UROD, PBGD,
or CPPO homologs, i.e. DNA sequences encoding ENR-A, CBL, UROD, PBGD, or CPPO enzymes,
respectively, are isolated from the genome of any desired plant.
[0061] Alternatively, ENR-A, CBL, UROD, PBGD, or CPPO gene sequences, can be isolated from
any plant according to well known techniques based on their sequence similarity to
the
Arabidopsis thaliana coding sequences (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9,
respectively) taught by the present invention. In these techniques, all or part of
a known ENR-A, CBL, UROD, PBGD, or CPPO gene's coding sequence, respectively, is used
as a probe that selectively hybridizes to other ENR-A, CBL, UROD, PBGD, or CPPO gene
sequences, present in a population of cloned genomic DNA fragments or cDNA fragments
(i.e. genomic or cDNA libraries) from a chosen source organism. Such techniques include
hybridization screening of plated DNA libraries (either plaques or colonies; see,
e.g.. Sambrook
et al., "Molecular Cloning", eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification
by PCR using oligonucleotide primers corresponding to sequence domains conserved among
known ENR-A, CBL, UROD, PBGD, or CPPO enzyme's amino acid sequences, respectively
(see, e.g. Innis
et al., "PCR Protocols, a Guide to Methods and Applications", Academic Press (1990)). These
methods are particularly well suited to the isolation of ENR-A, CBL, UROD, PBGD, or
CPPO gene sequences, from organisms closely related to the organism from which the
probe sequence is derived. The application of these methods using the
Arabidopsis coding sequences as probes is well suited for the isolation of ENR-A, CBL, UROD,
PBGD, or CPPO gene sequences, from any source organism, preferably other plant species,
including monocotyledons and dicotyledons.
[0062] The isolated ENR-A, CBL, UROD, PBGD, or CPPO gene sequences, taught by the present
invention can be manipulated according to standard genetic engineering techniques
to suit any desired purpose. For example, an entire plant ENR-A, CBL, UROD, PBGD,
or CPPO gene sequence, or portions thereof may be used as a probe capable of specifically
hybridizing to coding sequences and messenger RNAs. To achieve specific hybridization
under a variety of conditions, such probes include, e.g. sequences that are unique
among plant ENR-A, CBL, UROD, PBGD, or CPPO gene sequences, and are at least 10 nucleotides
in length, preferably at least 20 nucleotides in length, and most preferably at least
50 nucleotides in length. Such probes are used to amplify and analyze ENR-A, CBL,
UROD, PBGD, or CPPO gene sequences, respectively, from a chosen organism via PCR.
This technique is useful to isolate additional plant ENR-A, CBL, UROD, PBGD, or CPPO
gene sequences, respectively, from a desired organism or as a diagnostic assay to
determine the presence of ENR-A, CBL, UROD, PBGD, or CPPO gene sequences, in an organism.
This technique also is used to detect the presence of altered ENR-A, CBL, UROD, PBGD,
or CPPO gene sequences, associated with a particular condition of interest such as
herbicide tolerance, poor health, etc.
[0063] ENR-A, CBL, UROD, PBGD, or CPPO, specific hybridization probes also are used to map
the location of these native genes in the genome of a chosen plant using standard
techniques based on the selective hybridization of the probe to genomic sequences.
These techniques include, but are not limited to, identification of DNA polymorphisms
identified or contained within the probe sequence, and use of such polymorphisms to
follow segregation of the gene relative to other markers of known map position in
a mapping population derived from self fertilization of a hybrid of two polymorphic
parental lines (see e.g. Helentjaris
et al., Plant Mol. Biol.
5: 109 (1985); Sommer
et al. Biotechniques 12:82 (1992); D'Ovidio
et al., Plant Mol. Biol. 15: 169 (1990)). While any plant ENR-A, CBL, UROD, PBGD, or CPPO gene sequence, is contemplated
to be useful as a probe for mapping ENR-A, CBL, UROD, PBGD, or CPPO genes, respectively,
preferred probes are those gene sequences from plant species more closely related
to the chosen plant species, and most preferred probes are those gene sequences from
the chosen plant species. Mapping of ENR-A, CBL, UROD, PBGD, or CPPO genes, in this
manner is contemplated to be particularly useful for breeding purposes. For instance,
by knowing the genetic map position of a mutant ENR-A, CBL, UROD, PBGD, or CPPO gene,
that confers herbicide resistance, flanking DNA markers are identified from a reference
genetic map (see,
e.g., Helentjaris,
Trends Genet. 3: 217 (1987)). During introgression of the herbicide resistance trait into a new breeding
line, these markers are used to monitor the extent of linked flanking chromosomal
DNA still present in the recurrent parent after each round of back-crossing.
[0064] ENR-A, CBL, UROD, PBGD, or CPPO, specific hybridization probes also are used to quantify
levels of ENR-A, CBL, UROD, PBGD, or CPPO gene mRNA, respectively, in a plant using
standard techniques such as Northern blot analysis. This technique is useful as a
diagnostic assay to detect altered levels of ENR-A, CBL, UROD, PBGD, or CPPO gene
expression, respectively, that are associated with particular conditions such as enhanced
tolerance to herbicides that target ENR-A, CBL, UROD, PBGD, or CPPO genes.
II. Essentiality of ENR-A, CBL, UROD, PBGD, or CPPO Genes, in Plants Demonstrated
by Antisense Inhibition
[0065] As shown in the Examples below, the essentiality of ENR-A, CBL, UROD, PBGD, or CPPO
genes, for normal plant growth and development has been demonstrated by antisense
inhibition of expression of the ENR-A, CBL, UROD, PBGD, or CPPO gene, respectively,
in International patent application no. WO 99/27119 entitled "Method and Compositions
Useful for the Activation of Silent Transgenes", incorporated herein by reference.
In this system, a hybrid transcription factor gene is made that comprises a DNA-binding
domain and an activation domain. In addition, an activatable DNA construct is made
that comprises a synthetic promoter operatively linked to an activatable DNA sequence.
The hybrid transcription factor gene and synthetic promoter are selected such that
the DNA binding domain of the hybrid transcription factor is capable of binding specifically
to the synthetic promoter, which then activates expression of the activatable DNA
sequence. A first plant is transformed with the hybrid transcription factor gene,
and a second plant is transformed with the activatable DNA construct. The first plant
and second plants are crossed to produce a progeny plant containing both the sequence
encoding the hybrid transcription factor and the synthetic promoter, wherein the activatable
DNA sequence is expressed in the progeny plant. In the preferred embodiment, the activatable
DNA sequence is an antisense sequence capable of inactivating expression of ENR-A,
CBL, UROD, PBGD, or CPPO, respectively. Hence, the progeny plant will be unable to
normally express the endogenous gene.
[0066] This antisense validation system is especially useful for allowing expression of
traits that might otherwise be unrecoverable as constitutively driven transgenes.
For instance, foreign genes with potentially lethal effect or antisense genes or dominant-negative
mutations designed to abolish function of essential genes, while of great interest
in basic studies of plant biology, present inherent experimental problems. Decreased
transformation frequencies are often cited as evidence of lethality associated with
a particular constitutively driven transgene, but negative results of this type are
laden with alternative trivial explanations. The antisense validation system is described
in greater detail below:
A. Hybrid Transcription Factor Gene
[0067] A hybrid transcription factor gene for use in the antisense validation system described
herein comprises DNA sequences encoding (1) a DNA-binding domain and (2) an activation
domain that interacts with components of transcriptional machinery assembling at a
promoter.
B. Activatable DNA Construct
[0068] An activatable DNA construct for use in the antisense validation system described
herein comprises (1) a synthetic promoter operatively linked to (2) an activatable
DNA sequence. The synthetic promoter comprises at least one DNA binding site recognized
by the DNA binding domain of the hybrid transcription factor, and a minimal promoter,
preferably a TATA element derived from a promoter recognized by plant cells. More
particularly the TATA element is derived from a promoter recognized by the plant cell
type into which the synthetic promoter will be incorporated. Desirably, the DNA binding
site is repeated multiple times in the synthetic promoter so that the minimal promoter
may be more effectively activated, such that the activatable DNA sequence associated
with the synthetic promoter is more effectively expressed.
[0069] The activatable DNA sequence encompasses a DNA sequence, in this case ENR-A, CBL,
UROD, PBGD, or CPPO, for which stable introduction and expression in a plant cell
is desired. The activatable DNA sequence is operatively linked to the synthetic promoter
to form the activatable DNA construct. The activatable DNA sequence in the activatable
DNA construct is not expressed, i.e. is silent, in transgenic lines, unless a hybrid
transcription factor capable of binding to and activating the synthetic promoter,
is also present. The activatable DNA construct subsequently is introduced into cells,
tissues or plants to form stable transgenic lines expressing the activatable DNA sequence,
as described more fully below.
C. Transgenic Plants Containing the Hybrid Transcription Factor Gene or the Activatable
DNA Construct
[0070] The antisense validation system utilizes a first plant containing the hybrid transcription
factor gene and a second plant containing the activatable DNA construct. The hybrid
transcription factor genes and activatable DNA constructs described above are introduced
into the plants by methods well known and routinely used in the art, including but
not limited to crossing,
Agrobacterium-mediated transformation, protoplast transformation, Ti plasmid vectors, direct DNA
uptake such as microprojectile bombardment, liposome mediated uptake, micro-injection,
etc. Transformants are screened for the presence and functionality of the transgenes
according to standard methods known to those skilled in the art.
D. Transgenic Plants Containing Both the Hybrid Transcription Factor Gene and the
Activatable DNA Construct
[0071] F1 plants containing both the hybrid transcription factor gene and the activatable
DNA construct are generated by crossing said first and second plants and selected
for the presence of an appropriate marker. In contrast to plants containing the activatable
DNA construct alone, the F1 plants generate high levels of activatable DNA sequence
expression product. Expression of ENR-A, CBL, UROD, PBGD, or CPPO antisense molecules,
respectively, in such plants results in death or abnormal growth or development, indicating
that ENR-A, CBL, UROD, PBGD, or CPPO, respectively, is essential for normal plant
growth and development.
III. Recombinant Production of Plants ENR-A, CBL, UROD, PBGD, or CPPO Enzymes, and
Uses Thereof
[0072] For recombinant production of a plant ENR-A, CBL, UROD, PBGD, or CPPO enzyme, in
a host organism, a ENR-A, CBL, UROD, PBGD, or CPPO coding sequence, respectively,
preferably a plant coding sequence, is inserted into an expression cassette designed
for the chosen host and introduced into the host where it is recombinantly produced.
The choice of specific regulatory sequences such as promoter, signal sequence, 5'
and 3' untranslated sequences, and enhancer appropriate for the chosen host is within
the level of skill of the routineer in the art. The resultant molecule, containing
the individual elements operably linked in proper reading frame, is inserted into
a vector capable of being transformed into the host cell. Suitable expression vectors
and methods for recombinant production of proteins are well known for host organisms
such as
E. coli, yeast, and insect cells (see,
e.g., Luckow and Summers,
Bio/
Technol. 6: 47 (1988)). Specific examples include plasmids such as pBluescript (Stratagene,
La Jolla, CA; USA), pFLAG (International Biotechnologies, Inc., New Haven, CT, USA),
pTrcHis (Invitrogen, La Jolla, CA, USA), and baculovirus expression vectors, e.g.,
those derived from the genome of
Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculovirus/insect system is pVI11392/Sf21
cells (Invitrogen, La Jolla, CA, USA).
[0073] Recombinantly produced ENR-A, CBL, UROD, PBGD, or CPPO enzymes, respectively, are
isolated and purified using a variety of standard techniques. The actual techniques
used varies depending upon the host organism used, whether the enzyme is designed
for secretion, and other such factors. Such techniques are well known to the skilled
artisan (see,
e.g. chapter 16 of Ausubel, F.
et al., "Current Protocols in Molecular Biology", published by John Wiley & Sons, Inc. (1994).
[0074] Recombinantly produced ENR-A, CBL, UROD, PBGD, or CPPO enzymes are useful for a variety
of purposes. For example, they are used in
in vifro assays to screen known herbicidal chemicals, whose target has not been identified,
to determine if they inhibit ENR-A, CBL, UROD, PBGD, or CPPO enzymes, respectively.
Such
in vitro assays also are useful as screens to identify new chemicals that inhibit such enzymatic
activity and that are therefore novel herbicide candidates. Alternatively, recombinantly
produced ENR-A, CBL, UROD, PBGD, or CPPO enzymes, are used to further characterize
their association with known inhibitors in order to rationally design new inhibitory
herbicides as well as herbicide tolerant forms of the enzymes.
In Vitro Inhibitor Assays: Discovery of Small Molecule Ligand that Interacts with the Gene
Product of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9
[0075] Once a protein has been identified as a potential herbicide target, the next step
is to develop an assay that allows screening large number of chemicals to determine
which ones interact with the protein. Although it is straightforward to develop assays
for proteins of known function, developing assays with proteins of unknown functions
is more difficult. This difficulty can be overcome by using technologies that can
detect interactions between a protein and a compound without knowing the biological
function of the protein. A short description of three methods is presented, including
fluorescence correlation spectroscopy, surface-enhanced laser desorption/ionization,
and biacore technologies.
[0076] Fluorescence Correlation Spectroscopy (FCS) theory was developed in 1972 but it is
only in recent years that the technology to perform FCS became available (Madge et
al. (1972) Phys. Rev. Lett., 29: 705-708; Maiti et al. (1997) Proc. Natl. Acad. Sci.
USA, 94: 11753-11757). FCS measures the average diffusion rate of a fluorescent molecule
within a small sample volume. The sample size can be as low as 10
3 fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium.
The diffusion rate is a function of the mass of the molecule and decreases as the
mass increases. FCS can therefore be applied to protein-ligand interaction analysis
by measuring the change in mass and therefore in diffusion rate of a molecule upon
binding. In a typical experiment, the target to be analyzed is expressed as a recombinant
protein with a sequence tag, such as a poly-histidine sequence, inserted at the N
or C-terminus. The expression takes place in
E. coli, yeast or insect cells. The protein is purified by chromatography. For example, the
poly-histidine tag can be used to bind the expressed protein to a metal chelate column
such as Ni2+ chelated on iminodiacetic acid agarose. The protein is then labeled with
a fluorescent tag such as carboxytetramethylrhodamine or BODIPY® (Molecular Probes,
Eugene, OR, USA). The protein is then exposed in solution to the potential ligand,
and its diffusion rate is determined by FCS using instrumentation available from Carl
Zeiss, Inc. (Thornwood, NY, USA). Ligand binding is determined by changes in the diffusion
rate of the protein.
[0077] Surface-Enhanced Laser Desorption/lonization (SELDI) was invented by Hutchens and
Yip during the late 1980's (Hutchens and Yip (1993) Rapid Commun. Mass Spectrom. 7:
576-580). When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides
a mean to rapidly analyze molecules retained on a chip. It can be applied to ligand-protein
interaction analysis by covalently binding the target protein on the chip and analyze
by MS the small molecules that bind to this protein (Worrall et al. (1998) Anal. Biochem.
70: 750-756). In a typical experiment, the target to be analyzed is expressed as described
for FCS. The purified protein is then used in the assay without further preparation.
It is bound to the SELDI chip either by utilizing the poly-histidine tag or by other
interaction such as ion exchange or hydrophobic interaction. The chip thus prepared
is then exposed to the potential ligand via, for example, a delivery system capable
to pipet the ligands in a sequential manner (autosampler). The chip is then submitted
to washes of increasing stringency, for example a series of washes with buffer solutions
containing an increasing ionic strength. After each wash, the bound material is analyzed
by submitting the chip to SELDI-TOF. Ligands that specifically bind the target will
be identified by the stringency of the wash needed to elute them.
[0078] Biacore relies on changes in the refractive index at the surface layer upon binding
of a ligand to a protein immobilized on the layer. In this system, a collection of
small ligands is injected sequentially in a 2-5 microlitre cell with the immobilized
protein. Binding is detected by surface plasmon resonance (SPR) by recording laser
light refracting from the surface. In general, the refractive index change for a given
change of mass concentration at the surface layer, is practically the same for all
proteins and peptides, allowing a single method to be applicable for any protein (Liedberg
et al. (1983) Sensors Actuators 4: 299-304; Malmquist (1993) Nature, 361: 186-187).
In a typical experiment, the target to be analyzed is expressed as described for FCS.
The purified protein is then used in the assay without further preparation. It is
bound to the Biacore chip either by utilizing the poly-histidine tag or by other interaction
such as ion exchange or hydrophobic interaction. The chip thus prepared is then exposed
to the potential ligand via the delivery system incorporated in the instruments sold
by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler).
The SPR signal on the chip is recorded and changes in the refractive index indicate
an interaction between the immobilized target and the ligand. Analysis of the signal
kinetics on rate and off rate allows the discrimination between non-specific and specific
interaction.
[0079] Also, an assay for small molecule ligands that interact with a polypeptide is an
inhibitor assay. For example, such an inhibitor assay useful for identifying inhibitors
of essential plant genes, such as plant ENR-A, CBL, UROD, PBGD, or CPPO genes, comprises
the steps of:
a) reacting a plant ENR-A, CBL, UROD, PBGD, or CPPO enzyme, and a substrate thereof
in the presence of a suspected inhibitor of the enzyme's function;
b) comparing the rate of enzymatic activity in the presence of the suspected inhibitor
to the rate of enzymatic activity under the same conditions in the absence of the
suspected inhibitor; and
c) determining whether the suspected inhibitor inhibits the ENR-A, CBL, UROD, PBGD,
or CPPO enzyme, respectively.
[0080] For example, the inhibitory effect on plant ENR-A, CBL, UROD, PBGD, or CPPO, may
be determined by a reduction or complete inhibition of ENR-A, CBL, UROD, PBGD, or
CPPO activity, respectively, in the assay. Such a determination may be made by comparing,
in the presence and absence of the candidate inhibitor, the amount of substrate used
or intermediate or product made during the reaction.
IV. In Vivo Inhibitor Assay
[0081] In one embodiment, a suspected herbicide, for example identified by
in vitro screening, is applied to plants at various concentrations. The suspected herbicide
is preferably sprayed on the plants. After application of the suspected herbicide,
its effect on the plants, for example death or suppression of growth is recorded.
[0082] In another embodiment, an
in vivo screening assay for inhibitors of the ENR-A, CBL, UROD, PBGD, or CPPO activity, uses
transgenic plants, plant tissue, plant seeds or plant cells capable of overexpressing
a nucleotide sequence having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively,
wherein the ENR-A, CBL, UROD, PBGD, or CPPO gene product, is enzymatically active
in the transgenic plants, plant tissue, plant seeds or plant cells. The nucleotide
sequence is preferably derived from an eukaryote, such as a yeast, but is preferably
derived from a plant. In a further preferred embodiment, the nucleotide sequence is
identical or substantially similar to the nucleotide sequence set forth in SEO ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, or encodes an enzyme
having ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively, whose amino acid sequence
is identical or substantially similar to the amino acid sequence set forth in SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. In another preferred
embodiment, the nucleotide sequence is derived from a prokaryote.
[0083] A chemical is then applied to the transgenic plants, plant tissue, plant seeds or
plant cells and to the isogenic non-transgenic plants, plant tissue, plant seeds or
plant cells, and the growth or viability of the transgenic and non-transformed plants,
plant tissue, plant seeds or plant cells are determined after application of the chemical
and compared. Compounds capable of inhibiting the growth of the non-transgenic plants,
but not affecting the growth of the transgenic plants are selected as specific inhibitors
of ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively.
V. Herbicide Tolerant Plants
[0084] Development of tolerance can allow application of a herbicide to a crop where its
use was previously precluded or limited (
e.g. to pre-emergence use) due to sensitivity of the crop to the herbicide. For example,
U.S. Patent No. 4,761,373 to Anderson
et al. is directed to plants resistant to various imidazolinone or sulfonamide herbicides.
The resistance is conferred by an altered acetohydroxyacid synthase (AHAS) enzyme.
U.S. Patent No. 4,975,374 to Goodman
et al. relates to plant cells and plants containing a gene encoding a mutant glutamine
synthetase (GS) resistant to inhibition by herbicides that were known to inhibit GS,
e.g. phosphinothricin and methionine sulfoximine. U.S. Patent No. 5,013,659 to Bedbrook
et al. is directed to plants expressing a mutant acetolactate synthase that renders the
plants resistant to inhibition by sulfonylurea herbicides. U.S. Patent No. 5,162,602
to Somers
et al. discloses plants tolerant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic
acid herbicides. The tolerance is conferred by an altered acetyl coenzyme A carboxylase
(ACCase).
[0085] The present invention is further directed to plants, plant tissue, plant seeds, and
plant cells tolerant to herbicides that inhibit the naturally occurring ENR-A, CBL,
UROD, PBGD, or CPPO, in these plants, wherein the tolerance is conferred by altered
ENR-A, CBL, UROD, PBGD, or CPPO ENR-A enzyme activity, respectively. Altered ENR-A,
CBL, UROD, PBGD, or CPPO enzyme activity, is conferred upon a plant according to the
invention by increasing expression of wild-type herbicide-sensitive ENR-A, CBL, UROD,
PBGD, or CPPO enzyme, by providing additional wild-type ENR-A, CBL, UROD, PBGD, or
CPPO genes, to the plant, by expressing modified herbicide-tolerant ENR-A, CBL, UROD,
PBGD, or CPPO enzymes, in the plant, or by a combination of these techniques. Representative
plants include any plants to which these herbicides are applied for their normally
intended purpose. Preferred are agronomically important crops such as cotton, soybean,
oilseed rape, sugar beet, maize, rice, wheat, barley, oats, rye, sorghum, millet,
turf, forage, turf grasses, and the like.
A. Increased Expression of Wild-Type ENR-A, CBL, UROD, PBGD, or CPPO Enzymes
[0086] Achieving altered ENR-A, CBL, UROD, PBGD, or CPPO enzyme activity, through increased
expression results in a level of a ENR-A, CBL, UROD, PBGD, or CPPO enzyme, respectively,
in the plant cell at least sufficient to overcome growth inhibition caused by the
herbicide. The level of expressed enzyme generally is at least two times, preferably
at least five times, and more preferably at least ten times the natively expressed
amount. Increased expression is conferred in a number of ways, e.g., providing multiple
copies of a wild-type ENR-A, CBL, UROD, PBGD, or CPPO gene, respectively; multiple
occurrences of the coding sequence within the gene (
i.e. gene amplification) or a mutation in the non-coding, regulatory sequence of the
endogenous gene in the plant cell. Plants having such altered gene activity are obtained
by direct selection in plants by methods known in the art (see, e.g. U.S. Patent No.
5,162,602, and U.S. Patent No. 4,761,373, and references cited therein). These plants
also may be obtained by genetic engineering techniques known in the art. Increased
expression of a herbicide-sensitive ENR-A, CBL, UROD, PBGD, or CPPO gene, also is
accomplished by stably transforming a plant cell with a recombinant or chimeric DNA
molecule comprising a promoter capable of driving expression of an associated structural
gene in a plant cell operatively linked to a homologous or heterologous structural
gene encoding the ENR-A, CBL, UROD, PBGD, or CPPO enzyme.
B. Expression of Modified Herbicide-Tolerant ENR-A, CBL, UROD, PBGD, or CPPO Enzymes
[0087] According to this embodiment, plants, plant tissue, plant seeds, or plant cells are
stably transformed with a recombinant DNA molecule comprising a suitable promoter
functional in plants operatively linked to a coding sequence encoding a herbicide
tolerant form of a ENR-A, CBL, UROD, PBGD, or CPPO enzyme. A herbicide tolerant form
of the enzyme has at least one amino acid substitution, addition or deletion that
confers tolerance to an amount of a herbicide effective to inhibit the unmodified,
naturally occurring form of the ENR-A, CBL, UROD, PBGD, or CPPO enzyme. The transgenic
plants, plant tissue, plant seeds, or plant cells thus created are selected by conventional
selection techniques, whereby herbicide tolerant lines are isolated, characterized,
and developed. Below are described methods for obtaining genes that encode herbicide
tolerant forms of ENR-A, CBL, UROD, PBGD, or CPPO enzymes.
[0088] One strategy involves direct or indirect mutagenesis procedures on microbes. For
instance, a genetically manipulatable microbe such as
E. coli or
S. cerevisiae may be subjected to random mutagenesis
in vivo with mutagens such as UV light or ethyl or methyl methane sulfonate. Mutagenesis
procedures are described, for example, in Miller,
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA (1972); Davis
et al., Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA (1980); Sherman
et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA (1983); and U.S. Patent
No. 4,975,374. The microbe selected for mutagenesis contains a normal, inhibitor-sensitive
ENR-A, CBL, UROD, PBGD, or CPPO gene, and is dependent upon the activity conferred
by this gene. The mutagenized cells are grown in the presence of the inhibitor at
concentrations that inhibit the unmodified gene. Colonies of the mutagenized microbe
that grow better than the unmutagenized microbe in the presence of the inhibitor (i.e.
exhibit resistance to the inhibitor) are selected for further analysis. ENR-A, CBL,
UROD, PBGD, or CPPO genes, from these colonies are isolated, either by cloning or
by PCR amplification, and their sequences are elucidated. Sequences encoding altered
gene products are then cloned back into the microbe to confirm their ability to confer
inhibitor tolerance.
[0089] A method of obtaining mutant herbicide-tolerant alleles of a plant ENR-A, CBL, UROD,
PBGD, or CPPO gene, involves direct selection in plants. For example, the effect of
a mutagenized ENR-A, CBL, UROD, PBGD, or CPPO gene, on the growth inhibition of plants
such as
Arabidopsis, soybean, or maize is determined by plating seeds sterilized by art-recognized methods
on plates on a simple minimal salts medium containing increasing concentrations of
the inhibitor. Such concentrations are in the range of 0.001, 0.003, 0.01, 0.03, 0.1,
0.3, 1, 3, 10, 30, 110, 300, 1000 and 3000 parts per million (ppm). The lowest dose
at which significant growth inhibition can be reproducibly detected is used for subsequent
experiments.
[0090] Mutagenesis of plant material is utilized to increase the frequency at which resistant
alleles occur in the selected population. Mutagenized seed material is derived from
a variety of sources, including chemical or physical mutagenesis of seeds, or chemical
or physical mutagenesis of pollen (Neuffer, In
Maize for Biological Research Sheridan, ed. Univ. Press, Grand Forks, ND., pp. 61-64 (1982)), which is then used
to fertilize plants and the resulting M
1 mutant seeds collected. Typically for
Arabidopsis M
2 seeds, which are progeny seeds of plants grown from seeds mutagenized with chemicals,
such as ethyl methane sulfonate, or with physical agents, such as gamma rays or fast
neutrons, are plated at densities of up to 10,000 seeds/plate (10 cm diameter) on
minimal salts medium containing an appropriate concentration of inhibitor to select
for tolerance. Seedlings that continue to grow and remain green 7-21 days after plating
are transplanted to soil and grown to maturity and seed set. Progeny of these seeds
are tested for tolerance to a ENR-A, CBL, UROD, PBGD, or CPPO inhibitor. If the tolerance
trait is dominant, plants whose seed segregate 3:1 /resistant:sensitive are presumed
to have been heterozygous for the resistance at the M
2 generation. Plants that give rise to all resistant seed are presumed to have been
homozygous for the resistance at the M
2 generation. Such mutagenesis on intact seeds and screening of their M2 progeny seed
can also be carried out on other species, for instance soybean (see,
e.g. U.S. Pat. No. 5,084,082). Alternatively, mutant seeds to be screened for herbicide
tolerance are obtained as a result of fertilization with pollen mutagenized by chemical
or physical means.
[0091] Confirmation that the genetic basis of the herbicide tolerance is a modified ENR-A,
CBL, UROD, PBGD, or CPPO gene, is ascertained as exemplified below. First, alleles
of the ENR-A, CBL, UROD, PBGD, or CPPO gene, from plants exhibiting resistance to
the inhibitor are isolated using PCR with primers based either upon conserved regions
in the
Arabidopsis cDNA coding sequences shown in SEQ ID NO:1 or, more preferably, based upon the unaltered
ENR-A, CBL, UROD, PBGD, or CPPO gene sequence, from the plant used to generate tolerant
alleles. After sequencing the alleles to determine the presence of mutations in the
coding sequence, the alleles are tested for their ability to confer tolerance to the
inhibitor on plants into which the putative tolerance-conferring alleles have been
transformed. These plants are
Arabidopsis plants or any other plant whose growth is susceptible to the inhibitors. Second,
the ENR-A, CBL, UROD, PBGD, or CPPO genes, are mapped relative to known restriction
fragment length polymorphisms (RFLPs) (See, for example, Chang
et al. Proc. Natl. Acad, Sci, USA 85: 6856-6860 (1988); Nam
et al., Plant Cell 1: 699-705 (1989). The tolerance trait is independently mapped using the same markers.
When tolerance is due to a mutation in the ENR-A, CBL, UROD, PBGD, or CPPO gene, the
tolerance trait maps to a position indistinguishable from the position of the ENR-A,
CBL, UROD, PBGD, or CPPO gene.
[0092] Another method of obtaining herbicide-tolerant alleles of a ENR-A, CBL, UROD, PBGD,
or CPPO gene, is by selection in plant cell cultures. Explants of plant tissue,
e.g. embryos, leaf disks, etc. or actively growing callus or suspension cultures of a
plant of interest are grown on medium in the presence of increasing concentrations
of a ENR-A, CBL, UROD, PBGD, or CPPO, inhibitor. Varying degrees of growth are recorded
in different cultures. In certain cultures, fast-growing variant colonies arise that
continue to grow even in the presence of normally inhibitory concentrations of inhibitor.
The frequency with which such faster-growing variants occur can be increased by treatment
with a chemical or physical mutagen before exposing the tissues or cells to the inhibitor.
Putative tolerance-conferring alleles of the ENR-A, CBL, UROD, PBGD, or CPPO gene,
are isolated and tested as described in the foregoing paragraphs. Those alleles identified
as conferring herbicide tolerance may then be engineered for optimal expression and
transformed into the plant. Alternatively, plants can be regenerated from the tissue
or cell cultures containing these alleles.
[0093] Still another method involves mutagenesis of wild-type, herbicide sensitive plant
ENR-A, CBL, UROD, PBGD, or CPPO genes, in bacteria or yeast, followed by culturing
the microbe on medium that contains inhibitory concentrations of the inhibitor and
then selecting those colonies that grow in the presence of the inhibitor. More specifically,
a plant cDNA, such as the
Arabidopsis cDNA encoding ENR-A (SEQ ID NO:1), CBL (SEQ ID NO:3), UROD (SEQ ID NO:5), PBGD (SEQ
ID NO:7), or CPPO (SEQ ID NO:9), is cloned into a microbe that otherwise lacks the
selected gene's activity. The transformed microbe is then subjected to
in vivo mutagenesis or to
in vitro mutagenesis by any of several chemical or enzymatic methods known in the art, e.g.
sodium bisulfite (Shortle
et al., Methods Enzymol. 100:457-468 (1983); methoxylamine (Kadonaga
et al., Nucleic Acids Res. 13:1733-1745 (1985); oligonucleotide-directed saturation mutagenesis (Hutchinson
et al., Proc. Natl. Acad. Sci. USA, 83:710-714 (1986); or various polymerase misincorporation strategies (see, e.g. Shortle
et al., Proc. Natl. Acad. Sci. USA, 79:1588-1592 (1982); Shiraishi
et al., Gene 64:313-319 (1988); and Leung
et al., Technique 1:11-15 (1989). Colonies that grow in the presence of normally inhibitory concentrations
of inhibitor are picked and purified by repeated restreaking. Their plasmids are purified
and tested for the ability to confer tolerance to the inhibitor by retransforming
them into the microbe lacking ENR-A, CBL, UROD, PBGD, or CPPO gene activity. The DNA
sequences of cDNA inserts from plasmids that pass this test are then determined.
[0094] Herbicide resistant ENR-A, CBL, UROD, PBGD, or CPPO proteins, are also obtained using
methods involving
in vitro recombination, also called DNA shuffling. By DNA shuffling, mutations, preferably
random mutations, are introduced into nucleotide sequences encoding ENR-A, CBL, UROD,
PBGD, or CPPO activity, respectively. DNA shuffling also leads to the recombination
and rearrangement of sequences within a ENR-A, CBL, UROD, PBGD, or CPPO gene, or to
recombination and exchange of sequences between two or more different of ENR-A, CBL,
UROD, PBGD, or CPPO genes, respectively. These methods allow for the production of
millions of mutated ENR-A, CBL, UROD, PBGD, or CPPO coding sequences. The mutated
genes, or shuffled genes, are screened for desirable properties, e.g. improved tolerance
to herbicides and for mutations that provide broad spectrum tolerance to the different
classes of inhibitor chemistry. Such screens are well within the skills of a routineer
in the art.
[0095] In a preferred embodiment, a mutagenized ENR-A, CBL, UROD, PBGD, or CPPO gene, is
formed from at least one template ENR-A, CBL, UROD, PBGD, or CPPO gene, wherein the
template ENR-A, CBL, UROD, PBGD, or CPPO gene, has been cleaved into double-stranded
random fragments of a desired size, and comprising the steps of adding to the resultant
population of double-stranded random fragments one or more single or double-stranded
oligonucleotides, wherein said oligonucleotides comprise an area of identity and an
area of heterology to the double-stranded random fragments; denaturing the resultant
mixture of double-stranded random fragments and oligonucleotides into single-stranded
fragments; incubating the resultant population of single-stranded fragments with a
polymerase under conditions which result in the annealing of said single-stranded
fragments at said areas of identity to form pairs of annealed fragments, said areas
of identity being sufficient for one member of a pair to prime replication of the
other, thereby forming a mutagenized double-stranded polynucleotide; and repeating
the second and third steps for at least two further cycles, wherein the resultant
mixture in the second step of a further cycle includes the mutagenized double-stranded
polynucleotide from the third step of the previous cycle, and the further cycle forms
a further mutagenized double-stranded polynucleotide, wherein the mutagenized polynucleotide
is a mutated ENR-A, CBL, UROD, PBGD, or CPPO gene, having enhanced tolerance to a
herbicide which inhibits naturally occurring ENR-A, CBL, UROD, PBGD, or CPPO activity.
In a preferred embodiment, the concentration of a single species of double-stranded
random fragment in the population of double-stranded random fragments is less than
1% by weight of the total DNA. In a further preferred embodiment, the template double-stranded
polynucleotide comprises at least about 100 species of polynucleotides. In another
preferred embodiment, the size of the double-stranded random fragments is from about
5 bp to 5 kb. In a further preferred embodiment, the fourth step of the method comprises
repeating the second and the third steps for at least 10 cycles. Such method is described
e.g. in Stemmer et al. (1994) Nature 370: 389-391, in US Patent 5,605,793, US Patent
5,811,238 and in Crameri et al. (1998) Nature 391: 288-291, as well as in WO 97/20078,
and these references are incorporated herein by reference.
[0096] In another preferred embodiment, any combination of two or more different ENR-A,
CBL, UROD, PBGD, or CPPO genes, are mutagenized
in vitro by a staggered extension process (StEP), as described e.g. in Zhao et al. (1998)
Nature Biotechnology 16: 258-261. The two or more ENR-A, CBL, UROD, PBGD, or CPPO
genes, respectively, are used as template for PCR amplification with the extension
cycles of the PCR reaction preferably carried out at a lower temperature than the
optimal polymerization temperature of the polymerase. For example, when a thermostable
polymerase with an optimal temperature of approximately 72°C is used, the temperature
for the extension reaction is desirably below 72°C, more desirably below 65°C, preferably
below 60°C, more preferably the temperature for the extension reaction is 55°C. Additionally,
the duration of the extension reaction of the PCR cycles is desirably shorter than
usually carried out in the art, more desirably it is less than 30 seconds, preferably
it is less than 15 seconds, more preferably the duration of the extension reaction
is 5 seconds. Only a short DNA fragment is polymerized in each extension reaction,
allowing template switch of the extension products between the starting DNA molecules
after each cycle of denaturation and annealing, thereby generating diversity among
the extension products. The optimal number of cycles in the PCR reaction depends on
the length of the ENR-A, CBL, UROD, PBGD, or CPPO genes, to be mutagenized but desirably
over 40 cycles, more desirably over 60 cycles, preferably over 80 cycles are used.
Optimal extension conditions and the optimal number of PCR cycles for every combination
of ENR-A, CBL, UROD, PBGD, or CPPO genes, are determined as described in using procedures
well-known in the art. The other parameters for the PCR reaction are essentially the
same as commonly used in the art. The primers for the amplification reaction are preferably
designed to anneal to DNA sequences located outside of the ENR-A, CBL, UROD, PBGD,
or CPPO genes, e.g. to DNA sequences of a vector comprising the ENR-A, CBL, UROD,
PBGD, or CPPO genes, whereby the different ENR-A, CBL, UROD, PBGD, or CPPO genes,
used in the PCR reaction are preferably comprised in separate vectors. The primers
desirably anneal to sequences located less than 500 bp away from ENR-A, CBL, UROD,
PBGD, or CPPO sequences, preferably less than 200 bp away from the ENR-A, CBL, UROD,
PBGD, or CPPO sequences, more preferably less than 120 bp away from the ENR-A, CBL,
UROD, PBGD, or CPPO sequences. Preferably, the ENR-A, CBL, UROD, PBGD, or CPPO sequences,
are surrounded by restriction sites, which are included in the DNA sequence amplified
during the PCR reaction, thereby facilitating the cloning of the amplified products
into a suitable vector. In another preferred embodiment, fragments of ENR-A, CBL,
UROD, PBGD, or CPPO genes, having cohesive ends are produced as described in WO 98/05765.
The cohesive ends are produced by ligating a first oligonucleotide corresponding to
a part of a ENR-A, CBL, UROD, PBGD, or CPPO gene, to a second oligonucleotide not
present in the gene or corresponding to a part of the gene not adjoining to the part
of the gene corresponding to the first oligonucleotide, wherein the second oligonucleotide
contains at least one ribonucleotide. A double-stranded DNA is produced using the
first oligonucleotide as template and the second oligonucleotide as primer. The ribonucleotide
is cleaved and removed. The nucleotide(s) located 5' to the ribonucleotide is also
removed, resulting in double-stranded fragments having cohesive ends. Such fragments
are randomly reassembled by ligation to obtain novel combinations of gene sequences.
[0097] Any ENR-A, CBL, UROD, PBGD, or CPPO gene, or any combination of ENR-A, CBL, UROD,
PBGD, or CPPO genes, or homologs thereof, is used for
in vitro recombination in the context of the present invention, for example, a ENR-A, CBL,
UROD, PBGD, or CPPO gene, derived from a plant, such as, e.g.
Arabidopsis thaliana, e.g. a ENR-A gene set forth in SEQ ID NO:1, CBL gene set forth in SEQ ID NO:3, UROD
gene set forth in SEQ ID NO:5, PBGD gene set forth in SEQ ID NO:7, or CPPO gene set
forth in SEQ ID NO:9. Whole ENR-A, CBL, UROD, PBGD, or CPPO genes, or portions thereof
are used in the context of the present invention. The library of mutated ENR-A, CBL,
UROD, PBGD, or CPPO genes, obtained by the methods described above are cloned into
appropriate expression vectors and the resulting vectors are transformed into an appropriate
host, for example a plant cell, an algae like
Chlamydomonas, a yeast or a bacteria. An appropriate host requires ENR-A, CBL, UROD, PBGD, or CPPO
gene product activity, for growth. Host cells transformed with the vectors comprising
the library of mutated ENR-A, CBL, UROD, PBGD, or CPPO genes, are cultured on medium
that contains inhibitory concentrations of the inhibitor and those colonies that grow
in the presence of the inhibitor are selected. Colonies that grow in the presence
of normally inhibitory concentrations of inhibitor are picked and purified by repeated
restreaking. Their plasmids are purified and the DNA sequences of cDNA inserts from
plasmids that pass this test are then determined.
[0098] An assay for identifying a modified ENR-A, CBL, UROD, PBGD, or CPPO gene, that is
tolerant to an inhibitor may be performed in the same manner as the assay to identify
inhibitors of the ENR-A, CBL, UROD, PBGD, or CPPO enzyme, respectively, (Inhibitor
Assay, above) with the following modifications: First, a mutant ENR-A, CBL, UROD,
PBGD, or CPPO enzyme, is substituted in one of the reaction mixtures for the wild-type
ENR-A, CBL, UROD, PBGD, or CPPO enzyme, respectively, of the inhibitor assay. Second,
an inhibitor of wild-type enzyme is present in both reaction mixtures. Third, mutated
activity (activity in the presence of inhibitor and mutated enzyme) and unmutated
activity (activity in the presence of inhibitor and wild-type enzyme) are compared
to determine whether a significant increase in enzymatic activity is observed in the
mutated activity when compared to the unmutated activity. Mutated activity is any
measure of activity of the mutated enzyme while in the presence of a suitable substrate
and the inhibitor. Unmutated activity is any measure of activity of the wild-type
enzyme while in the presence of a suitable substrate and the inhibitor. A significant
increase is defined as an increase in enzymatic activity that is larger than the margin
of error inherent in the measurement technique, preferably an increase by about 2-fold
or greater of the activity of the wild-type enzyme in the presence of the inhibitor,
more preferably an increase by about 5-fold or greater, most preferably an increase
by about 10-fold or greater.
[0099] In addition to being used to create herbicide-tolerant plants, genes encoding herbicide
tolerant ENR-A, CBL, UROD, PBGD, or CPPO enzymes, also are used as selectable markers
in plant cell transformation methods. For example, plants, plant tissue, plant seeds,
or plant cells transformed with a transgene are transformed with a gene encoding an
altered ENR-A, CBL, UROD, PBGD, or CPPO enzyme, capable of being expressed by the
plant. The transformed cells are transferred to medium containing an ENR-A, CBL, UROD,
PBGD, or CPPO inhibitor, in an amount sufficient to inhibit the survivability of plant
cells not expressing the modified gene, wherein only the transformed cells will survive.
The method is applicable to any plant cell capable of being transformed with a modified
ENR-A, CBL, UROD, PBGD, or CPPO enzyme-encoding gene, and can be used with any transgene
of interest. Expression of the transgene and the inhibitor-tolerant CBL, UROD, PBGD,
CPPO, or ENR-A gene, can be driven by the same promoter functional in plant cells,
or by separate promoters.
VI. Plant Transformation Technology
[0100] A wild-type or herbicide-tolerant form of the ENR-A, CBL, UROD, PBGD, or CPPO gene,
can be incorporated in plant or bacterial cells using conventional recombinant DNA
technology. Generally, this involves inserting a DNA molecule encoding the ENR-A,
CBL, UROD, PBGD, or CPPO enzyme, into an expression system to which the DNA molecule
is heterologous (i.e., not normally present) using standard cloning procedures known
in the art. The vector contains the necessary elements for the transcription and translation
of the inserted protein-coding sequences in a host cell containing the vector. A large
number of vector systems known in the art can be used, such as plasmids, bacteriophage
viruses and other modified viruses. The components of the expression system optionally
are modified to increase expression. For example, truncated sequences, nucleotide
substitutions or other modifications optionally are employed. Expression systems known
in the art are used to transform virtually any crop plant cell under suitable conditions.
Transformed cells are regenerated into whole plants such that the chosen form of the
ENR-A, CBL, UROD, PBGD, or CPPO gene, confers herbicide tolerance in the transgenic
plants.
A. Requirements for Construction of Plant Expression Cassettes
[0101] Gene sequences intended for expression in transgenic plants are first operably linked
to a suitable promoter expressible in plants. Such expression cassettes optionally
comprise further sequences required or selected for the expression of the transgene.
Such sequences include, but are not restricted to, transcription terminators, extraneous
sequences to enhance expression such as introns, vital sequences, and sequences intended
for the targeting of the gene product to specific organelles and cell compartments.
These expression cassettes are easily transferred to the plant transformation vectors
described
infra. The following is a description of various components of typical expression cassettes.
1. Promoters
[0102] The selection of the promoter used determines the spatial and temporal expression
pattern of the transgene in the transgenic plant. Selected promoters will express
transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells,
root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for
example) and the selection will reflect the desired location of accumulation of the
gene product. Alternatively, the selected promoter may drive expression of the gene
under various inducing conditions. Promoters vary in their strength, i.e., ability
to promote transcription. Depending upon the host cell system utilized, any one of
a number of suitable promoters known in the art can be used. For example, for constitutive
expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter
may be used. For regulatable expression, the chemically inducible PR-1 promoter from
tobacco or
Arabidopsis may be used (
see, e.g., U.S. Patent No. 5,689,044).
2. Transcriptional Terminators
[0103] A variety of transcriptional terminators are available for use in expression cassettes.
These are responsible for the termination of transcription beyond the transgene and
its correct polyadenylation. Appropriate transcriptional terminators are those that
are known to function in plants and include the CaMV 35S terminator, the
tml terminator, the nopaline synthase terminator and the pea
rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
3. Sequences for the Enhancement or Regulation of Expression
[0104] Numerous sequences are known to enhance gene expression from within the transcriptional
unit and these sequences can be used in conjunction with the genes of this invention
to increase their expression in transgenic plants. For example, various intron sequences
such as introns of the maize
Adhl gene have been shown to enhance expression, particularly in monocotyledonous cells.
In addition, a number of non-translated leader sequences derived from viruses also
are known to enhance expression, and these are particularly effective in dicotyledonous
cells.
4. Coding Sequence Optimization
[0105] The coding sequence of the selected gene optionally is genetically engineered by
altering the coding sequence for optimal expression in the crop species of interest.
Methods for modifying coding sequences to achieve optimal expression in a particular
crop species are well known (see,
e.g. Perlak
et al., Proc. Natl. Acad. Sci. USA 88: 3324 (1991); and Koziel
et al., Bio/
technol. 11: 194 (1993); Fennoy and Bailey-Serres.
Nucl. Acids Res. 21: 5294-5300 (1993). Methods for modifying coding sequences by taking into account
codon usage in plant genes and in higher plants, green algae, and cyanobacteria are
well known (see table 4 in: Murray et al.
Nucl. Acids Res. 17: 477-498 (1989); Campbell and Gowri
Plant Physiol. 92: 1-11(1990).
5. Targeting of the Gene Product Within the Cell
[0106] Various mechanisms for targeting gene products are known to exist in plants and the
sequences controlling the functioning of these mechanisms have been characterized
in some detail. For example, the targeting of gene products to the chloroplast is
controlled by a signal sequence found at the amino terminal end of various proteins
which is cleaved during chloroplast import to yield the mature protein (
e.g. Comai
et al. J. Biol. Chem.
263: 15104-15109 (1988)). Other gene products are localized to other organelles such
as the mitochondrion and the peroxisome (
e.g. Unger
et al. Plant Molec. Biol.
13: 411-418 (1989)). The cDNAs encoding these products are manipulated to effect the
targeting of heterologous gene products to these organelles. In addition, sequences
have been characterized which cause the targeting of gene products to other cell compartments.
Amino terminal sequences are responsible for targeting to the ER, the apoplast, and
extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell
2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy
terminal sequences are responsible for vacuolar targeting of gene products (Shinshi
et al. Plant Molec. Biol.
14: 357-368 (1990)). By the fusion of the appropriate targeting sequences described
above to transgene sequences of interest one skilled in the art is able to direct
the transgene product to any organelle or cell compartment.
B. Construction of Plant Transformation Vectors
[0107] Numerous transformation vectors available for plant transformation are known to those
of ordinary skill in the plant transformation arts, and the genes pertinent to this
invention are used in conjunction with any such vectors. The selection of vector will
depend upon the preferred transformation technique and the target species for transformation.
For certain target species, different antibiotic or herbicide selection markers may
be preferred. Selection markers used routinely in transformation include the
nptll gene, which confers resistance to kanamycin and related antibiotics (Vieira & Messing
Gene
19: 259-268 (1982); Bevan et al., Nature
304:184-187 (1983)), the
bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl.
Acids Res
18: 1062 (1990), Spencer et al. Theor. Appl. Genet
79: 625-631 (1990)), the
hph gene, which confers resistance to the antibiotic hygromycin (Blochlinger & Diggelmann,
Mol Cell Biol
4: 2929-2931), and the
dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J.
2(7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S.
Patent Nos. 4,940,935 and 5,188,642).
1. Vectors Suitable for Agrobacterium Transformation
[0108] Many vectors are available for transformation using
Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such
as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Typical vectors suitable for
Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001, as well as the binary
vector pCIB10 and hygromycin selection derivatives thereof. (
See, for example, U.S. Patent No. 5,639,949).
2. Vectors Suitable for non-Agrobacterium Transformation
[0109] Transformation without the use of
Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector
and consequently vectors lacking these sequences can be utilized in addition to vectors
such as the ones described above which contain T-DNA sequences. Transformation techniques
that do not rely on
Agrobacterium include transformation via particle bombardment, protoplast uptake (
e.g. PEG and electroporation) and microinjection. The choice of vector depends largely
on the preferred selection for the species being transformed. Typical vectors suitable
for non-
Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35. (
See, for example, U.S. Patent No. 5,639,949).
C. Transformation Techniques
[0110] Once the coding sequence of interest has been cloned into an expression system, it
is transformed into a plant cell. Methods for transformation and regeneration of plants
are well known in the art. For example, Ti plasmid vectors have been utilized for
the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation,
micro-injection, and microprojectiles. In addition, bacteria from the genus
Agrobacterium can be utilized to transform plant cells.
[0111] Transformation techniques for dicotyledons are well known in the art and include
Agrobacterium-based techniques and techniques that do not require
Agrobacterium. Non-
Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts
or cells. This can be accomplished by PEG or electroporation mediated uptake, particle
bombardment-mediated delivery, or microinjection. In each case the transformed cells
are regenerated to whole plants using standard techniques known in the art.
[0112] Transformation of most monocotyledon species has now also become routine. Preferred
techniques include direct gene transfer into protoplasts using PEG or electroporation
techniques, particle bombardment into callus tissue, as well as
Agrobacterium-mediated transformation.
D. Plastid Transformation
[0113] In another preferred embodiment, a nucleotide sequence encoding a polypeptide having
ENR-A, CBL, UROD, PBGD, or CPPO activity, is directly transformed into the plastid
genome. Plastid expression, in which genes are inserted by homologous recombination
into the several thousand copies of the circular plastid genome present in each plant
cell, takes advantage of the enormous copy number advantage over nuclear-expressed
genes to permit expression levels that can readily exceed 10% of the total soluble
plant protein. In a preferred embodiment, the nucleotide sequence is inserted into
a plastid targeting vector and transformed into the plastid genome of a desired plant
host. Plants homoplasmic for plastid genomes containing the nucleotide sequence are
obtained, and are preferentially capable of high expression of the nucleotide sequence.
[0114] Plastid transformation technology is for example extensively described in U.S. Patent
Nos. 5,451,513, 5,545,817, 5,545,818, and 5,877,462 in PCT application no. WO 95/16783
and WO 97/32977, and in McBride
et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305, all incorporated herein by reference
in their entirety. The basic technique for plastid transformation involves introducing
regions of cloned plastid DNA flanking a selectable marker together with the nucleotide
sequence into a suitable target tissue, e.g., using biolistics or protoplast transformation
(e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking
regions, termed targeting sequences, facilitate homologous recombination with the
plastid genome and thus allow the replacement or modification of specific regions
of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12
genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable
markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc.
Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell
4, 39-45). The presence of cloning sites between these markers allowed creation of
a plastid targeting vector for introduction of foreign genes (Staub, J.M., and Maliga,
P. (1993)
EMBO J. 12, 601-606). Substantial increases in transformation frequency are obtained by replacement
of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable
marker, the bacterial
aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase
(Svab, Z., and Maliga, P. (1993)
Proc. Natl. Acad. Sci. USA 90, 913-917). Other selectable markers useful for plastid transformation are known
in the art and encompassed within the scope of the invention.
VII. Breeding
[0115] The wild-type or altered form of a ENR-A, CBL, UROD, PBGD, or CPPO gene, of the present
invention is utilized to confer herbicide tolerance to a wide variety of plant cells,
including those of gymnosperms, monocots, and dicots. Although the gene can be inserted
into any plant cell falling within these broad classes, it is particularly useful
in crop plant cells, such as rice, wheat, barley, rye, corn, potato, carrot, sweet
potato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip,
radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash,
pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine,
apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango,
banana, soybean, tobacco, tomato, sorghum and sugarcane.
[0116] The high-level expression of a wild-type ENR-A, CBL, UROD, PBGD, or CPPO gene, and/or
the expression of herbicide-tolerant forms of a ENR-A, CBL, UROD, PBGD, or CPPO gene,
conferring herbicide tolerance in plants, in combination with other characteristics
important for production and quality, is incorporated into plant lines through breeding
approaches and techniques known in the art.
[0117] Where a herbicide tolerant ENR-A, CBL, UROD, PBGD, or CPPO gene allele, is obtained
by direct selection in a crop plant or plant cell culture from which a crop plant
can be regenerated, it is moved into commercial varieties using traditional breeding
techniques to develop a herbicide tolerant crop without the need for genetically engineering
the allele and transforming it into the plant.
[0118] The invention will be further described by reference to the following detailed examples.
These examples are provided for purposes of illustration only, and are not intended
to be limiting unless otherwise specified.
EXAMPLES
[0119] Standard recombinant DNA and molecular cloning techniques used here are well known
in the art and are described by Sambrook,
et al.,
Molecular Cloning, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and by
T.J. Silhavy, M.L. Berman, and L.W. Enquist,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY , USA(1984) and by Ausubel,
F.M.
et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).
Example 1: Regulation of the Expression of the CBL Gene
[0120] The CBL gene encodes a protein that carries out a step in the methionine biosynthesis
pathway. CBL catalyzes the conversion of cystathionine to homocysteine (reviewed in
Ravanel et al. (1998)
Proc. Natl. Acad, Sci, USA 95: 7805-7812). The sequence of a cDNA for the
Arabidopsis CBL gene has been identified (Ravanel et al. (1995)
Plant Mol. Biol. 29: 875-882). The effect of the regulation of its expression in plants is tested
using constructs for sense RNA expression (sense construct), antisense RNA expression
(antisense construct) and antisense and sense RNA expression (antisense/sense construct).
A. Antisense construct: binary BASTA vector pJG261 (Guyer et al, Genetics (1998), 149: 633-639) is used containing a fragment from the pJG304ΔXhoI vector (see
below) with an insertion of part of the CBL gene in an antisense orientation (nucleotides
#13-1159, GenBank accession #L40511).
pJG304ΔXhol: Plasmid pJG304 (Guyer et al, Genetics (1998), 149: 633-639) is partially digested with Asp718 to isolate a full-length linear fragment. This fragment is ligated with a molar
excess of the 22 base oligonucleotide JG-L (5' GTA CCT CGA GTC TAG ACT CGA G 3'; SEQ
ID NO:32). Restriction analysis is used to identify a clone with this linker inserted
5' to the GAL4 DNA binding site, and this plasmid is designated pJG304ΔXhoI.
pJG304/aCBL: Plasmid pJG304ΔXhoI is digested with NcoI and SacI to excise the GUS gene. The GUS gene from pJG304ΔXhoI is replaced with a CBL PCR
product also digested with NcoI and SacI. This product is generated using primers DG354 (5' GAT CGA GCT CCA CGA GAA CTG TCT
CCG 3'; SEQ ID NO:14) and DG357 (5' TCA GCC ATG GGA AGA CAA GTA CAT TGC 3'; SEQ ID
NO:15) and the pFL61 Arabidopsis cDNA library (Minet et al. (1992) Plant J. 2: 417-422) as a template. Plasmid pJG304/aCBL is constructed from the pJG304ΔXhoI-digested
vector ligated to the CBL PCR product.
pJG261/aCBL: pJG304/aCBL is cut with Xhol to excise the cassette containing the GAL4 DNA binding site/35S minimal promoter/antisense
CBL/CaMV terminator fusion. This cassette is ligated into XhoI-digested pJG261 (Guyer et al, Genetics (1998), 149: 633-639), producing pJG261/aCBL.
B. Sense construct: same as antisense construct, except the CBL fragment is in the
opposite orientation. This construct contains the ATG start codon and most of the
CBL ORF and serves as a control for regulation of the expression of the CBL gene.
pJG304/sCBL: Plasmid pJG304ΔXhoI is digested with NcoI and SacI to excise the GUS gene. The GUS gene from pJG304ΔXhoI is replaced with a CBL PCR
product also digested with NcoI and SacI. This product is generated using primers CBL1 (5' CTT GCC ATG GCA CGA GAA CTG TCT
CCG 3'; SEQ ID NO:8) and CBL2 (5' CAT GGA GCT CGA AGA CAA GTA CAT TGC A 3'; SEQ ID
NO:17) and the pFL61 Arabidopsis cDNA library as a template. Plasmid pJG304/sCBL is constructed from the pJG304ΔXhoI-digested
vector ligated to the CBL PCR product.
pJG261/sCBL: pJG304/sCBL is cut with Xhol to excise the cassette containing the GAL4 DNA binding site/35S minimal promoter/sense
CBL/CaMV terminator fusion. This cassette is ligated into Xhol-digested pJG261 (Guyer et al, Genetics (1998), 149: 633-639), producing pJG261/sCBL.
C. Antisense/sense construct: A CBL gene fragment (#13-1159, GenBank accession # L40511)
in the sense orientation is inserted into the Sa/l site of vector pJG304ΔXhol downstream
of the antisense orientation version of the CBL gene. A linker of about 10 bp is present
between the two copies of CBL.
pJG304/dsCBL: Plasmid pJG304/aCBL is digested with SacI. A CBL PCR product also digested with SacI is inserted so that the inserted CBL gene is in the sense orientation. This product
is generated using CBL2 (5' CAT GGA GCT CGA AGA CAA GTA CAT TGC A 3'; SEQ ID NO:17)
and CBL3 (5' CAT CGA GCT CCT CTG TTT AAA CCA CGA GAA CTG TCT CCG TCG C 3'; SEQ ID
NO:18) and the pFL61 Arabidopsis cDNA library as a template. The plasmid construct with the desired orientation of
the inserted DNA is identified by digestion with HindIII. Plasmid pJG304/dsCBL is constructed from the pJG304/aCBL-digested vector ligated
to the CBL PCR product. SURE2 (Stratagene, LaJolla, CA, USA) is used as the bacterial
host to stabilize the construct.
pJG261/dsCBL: pJG304/dsCBL is cut with Xbal to excise the cassette containing the GAL4 DNA binding site/35S minimal promoter/antisense
CBL/sense CBL/CaMV terminator fusion. This cassette is ligated into SpeI-digested pJG261 (Guyer et al, Genetics (1998), 149: 633-639), producing pJG261/dsCBL. XL1-BLUE MRF' (Stratagene, LaJolla,
CA, USA) is used as the bacterial host to partially stabilize the construct. Unrearranged
DNA for this construct is isolated by agarose gel purification.
D. Production of GAL4 Binding Site/Minimal CaMV 35S/CBL Transgenic Plants
The three described pJG261/CBL constructs are electro-transformed (Bio-Rad Laboratories,
Hercules, CA) into Agrobacterium tumefaciens recA- strain AGL1 (Lazo et al. (1991) Bio/Technology 9: 963-967), and Arabidopsis plants (Ecotype Columbia) are transformed by infiltration (Bechtold et al., (1993)
C. R. Acad. Sci. Paris, 316: 1188-1193). Seeds from the infiltrated plants are selected on germination medium
(Murashige-Skoog salts at 4.3 g/liter, Mes at 0.5 g/liter, 1% sucrose, thiamine at
10 µg/liter, pyridoxine at 5 µg/liter, nicotinic acid at 5 µg/liter, myo-inositol
at 1 mg/liter, pH 5.8) containing Basta at 15 mg/liter.
E. Comparison of the Inhibition of CBL Using a GAL4/C1 Transactivator and a GAL4 Binding
Site/Minimal 35S Promoter
Transgenic plants containing a GAL4 binding site/minimal CaMV 35S promoter/ CBL construct
are transplanted to soil and grown to maturity in the greenhouse. The presence of
a transgenic CBL fragment in each line is confirmed by PCR. To test for the antisense
construct, primers ASV1 (5' TTT GGA GAG GAC AGA CCT GC 3'; SEQ ID NO:19) and CBL3
(5' CAT CGA GCT CCT CTG TTT AAA CCA CGA GAA CTG TCT CCG TCG C 3'; SEQ ID NO:18) are
used to verify the presence of an approximately 1200 bp product. Six transgenic lines
with the antisense construct are identified. To test for the sense construct, primers
ASV2 (5' GGA TTT TGG TTT TAG GAA TTA GAA 3'; SEQ ID NO:20) and CBL3 (5' CAT CGA GCT
CCT CTG TTT AAA CCA CGA GAA CTG TCT CCG TCG C 3'; SEQ ID NO:18) are used to verify
the presence of an approximately 1200 bp product. Thirteen transgenic lines with the
sense construct are identified. To test for the antisense/sense construct, primers
ASV2 (5' GGA TTT TGG TTT TAG GAA TTA GAA 3'; SEQ ID NO:20) and CBL3 (5' CAT CGA GCT
CCT CTG TTT AAA CCA CGA GAA CTG TCT CCG TCG C 3'; SEQ ID NO:18) are used to verify
the presence of an approximately 1200 bp product. In addition, to test for the antisense/sense
construct, primers ASV1 (5' TTT GGA GAG GAC AGA CCT GC 3'; SEQ ID NO:19) and CBL3
(5' CAT CGA GCT CCT CTG TTT AAA CCA CGA GAA CTG TCT CCG TCG C 3'; SEQ ID NO:18) are
used to verify the presence of an approximately 1200 bp product. Eleven transgenic
lines with the antisense/sense construct are identified.
Flowers borne on the primary transformants are crossed to pollen from the homozygous
GAL4/C1 transactivator line pAT53-103 (Guyer et al, Genetics (1998) 149: 633-649). F1 seeds are plated on MS + 2% sucrose medium (Murashige-Skoog
salts at 4.3 g/liter, Mes at 0.5 g/liter, 2% sucrose). None of the lines comprising
the antisense construct show an abnormal phenotype for the F1 progeny on plates. Two
of thirteen lines comprising the sense construct show a weak phenotype for approximately
half of the F1 progeny on each plate. The other eleven of thirteen lines comprising
the sense construct do not show an abnormal phenotype for the F1 progeny on plates.
Ten of eleven lines comprising the antisense/sense construct show phenotypes ranging
from weak to strong for approximately half of the F1 progeny on each plate. Plants
with a strong phenotype do not survive and have an increase in purple coloration,
lose green pigmentation, and fail to form leaves after fourteen days on the plates.
Plants with weaker phenotypes have some purple coloration, are paler green than normal,
and form smaller leaves after fourteen days on the plates. Thus, the inventors are
the first to demonstrate that the CBL gene is essential for the growth of a plant.
Previously, it has been shown that tobacco mutants lacking CBL activity were unable
to grow without exogenously-supplied methionine (Negrutiu et al. (1985) Mol. Gen. Genet. 199: 330-337), but the molecular nature of the mutation has not been shown.
Example 2: Isolation of a cDNA Encoding UROD from Arabidopsis
[0121] Primers UROD-N-Nde (5'-GGGTTTCCATATGTCAATCCTTCAAGTCTC-3'; SEQ ID NO:22) and UROD-C-Not
(5'-TTGCGCGGCCGCTTAATATCTAATTTCTTGAGC-3'; SEQ ID NO:23) are designed to the 5' and
3' ends of the predicted UROD ORF (Open Reading Frame) from BAC genomic sequence (GenBank
accession # AC002336), and PCR is performed using DNA from the pFL61
Arabidopsis Landsberg cDNA library (Minet et al. (1992)
Plant J. 2: 417-422) as the template. Another RT-PCR is also performed using RNA isolated
from
Arabidopsis Col-0 leaf tissue. The resulting PCR products are digested with
Ndel and
Notl and ligated to pET 32a vector DNA (Stratagene, LaJolla, CA, USA) treated with the
same restriction enzymes and sequenced. The UROD sequences from Col-0 and Landsberg
are the same. Both are identical with the predicted ORF. The prior indicated exon/intron
boundaries are: 48272..48787, 48874..48999, 49107..49295, 49391..49501, 49603..49727,
50182..50299, in the current version of GenBank accession # AC002336 annotated as
36805..36922, 37377..37501, 37603..37713, 37809..37997, 38105..38230, 38317..38832.
The cDNA sequence is the same as the sequence predicted in the GenBank annotation,
thus validating for the first time the putative open reading frame annotation.
[0122] The
Arabidopsis cDNA sequence encoding the UROD ORF is set forth in SEQ ID NO:5 and the encoded amino
acid sequence is set forth in SEQ ID NO:6.
Example 3: Construction of a Vector Containing a GAL4 Binding Site/Minimal 35S CaMV
Promoter Fused to Antisense UROD
[0123] pJG304/UD: Plasmid pJG304ΔXhoI (Guyer et al,
Genetics (1998), 149: 633-639) is digested with
Ncol and
BglII to excise the GUS gene. The GUS gene from pJG304ΔXhol is replaced with a UROD PCR
product digested with
AflIII (compatible with
Ncol) and
BamHI (compatible with
BglII). This product is generated using primers UROD-F2 (5'-CCCGGATCCATGTCAATCCTTCAAGTC-3';
SEQ ID NO:24) and UROD-R2 (5'-CCCACATGTATATCTAATTTCTTGAGC-3'; SEQ ID NO:25) and the
pFL61 cDNA library as a template. Plasmid pJG304/UD is constructed from the pJG304ΔXhol
digested vector ligated to the UROD PCR product.
Example 4: Plant Transformation Vectors for UROD Antisense Expression from the GAL4
Binding Site/CaMV Minimal 35S Promoter
[0124] pJG261/UD: pJG304/UD is cut with
Xhol to excise the cassette containing the GAL4 DNA binding site/35S minimal promoter/antisense
UROD/CaMV terminator fusion. This cassette is ligated into
XhoI-digested pJG261 (Guyer et al;
Genetics (1998), 149:633-639), producing pJG261/UD.
Example 5: Production Of GAL4 Binding Site/Minimal CaMV 35S Antisense UROD Transgenic
Plants
[0125] pJG261/UD is electro-transformed (Bio-Rad Laboratories, Hercules, CA) into
Agrobacterium tumefaciens strain GV3101, and
Arabidopsis plants (Ecotype Columbia) are transformed by infiltration (Bechtold,
et al., (1993)
C. R. Acad. Sci. Paris, 316: 1188-93). Seeds from the infiltrated plants are selected on germination medium
(Murashige-Skoog salts at 4.3 g/liter, Mes at 0.5 g/liter, 1% sucrose, thiamine at
10 µg/liter, pyridoxine at 5 µg/liter, nicotinic acid at 5 µg/liter, myo-inositol
at 1 mg/liter, pH 5.8) containing Basta at 15 mg/liter.
Example 6: Antisense Inhibition of UROD Using a GAL4/C1 Transactivator and a GAL4
Binding Site/Minimal CaMV 35S Promoter
[0126] Fifteen transgenic plants containing the GAL4 binding site/minimal CaMV 35S promoter/antisense
UROD construct are transplanted to soil and grown to maturity in the greenhouse. Flowers
borne on the primary transformants are crossed to pollen from the homozygous GAL4/C1
transactivator line pAT53-103 (Guyer et al,
Genetics (1998) 149:633-649). F1 seeds are plated on MS + 2% sucrose medium (Murashige-Skoog
salts at 4.3 g/liter, Mes at 0.5 g/liter, 2% sucrose) 6 lines segregate about 50%
seedlings with a bleached lethal phenotype on plates. Thus, the inventors are the
first to demonstrate that the UROD gene is essential for the growth of a dicot. Previously,
it has been shown that maize plants homozygous for a loss-of-function mutation in
this gene are dead (Hu et al,
Plant Cell (1998) 10:1095-1105). In addition, it has been shown that tobacco plants, expressing
a transgenic antisense construct, with 45% residual UROD activity exhibit necrosis,
but not lethality (Mock et al,
Plant Physiol. (1997) 113:1101-1112).
Example 7: Construction of a Vector Containing a GAL4 Binding Site/Minimal 35S CaMV
Promoter Fused to Antisense PBGD
[0127] pJG304/UD: Plasmid pJG304ΔXhoI (Guyer et al,
Genetics (1998), 149: 633-639) is digested with
NcoI and
BglII to excise the GUS gene. The GUS gene from pJG304ΔXhoI is replaced with a PBGD PCR
product digested with
BspHI (compatible with
NcoI) and
BglII. This product is generated using primers PORD-F2 (5'-CCC AGA TCT CCA TGG ATA TTG
CTT CGT C-3'; SEQ ID NO:27) and PORD-R2 (5'-CCC TCA TGA AGA TAG CAA TTC TTG CCC-3';
SEQ ID NO:28) and the pFL61
Arabidopsis cDNA library (Minet et al. (1992) Plant J. 2: 417-422) as a template. Plasmid pJG304/PD
is constructed from the pJG304ΔXhol digested vector ligated to the PBGD PCR product.
Example 8: Plant Transformation Vectors for PBGD Antisense Expression from the GAL4
Binding Site/CaMV Minimal 35S Promoter
[0128] pJG261/PD: pJG304/PD is cut with Xhol to excise the cassette containing the GAL4
DNA binding site/35S minimal promoter/antisense PBGD/CaMV terminator fusion. This
cassette is ligated into
XhoI-digested pJG261 (Guyer et al,
Genetics (1998), 149:633-639), producing pJG261/PD.
Example 9: Production of GAL4 Binding Site/Minimal CaMV 35S Antisense PBGD Transgenic
Plants
[0129] pJG261/PD is electro-transformed (Bio-Rad Laboratories, Hercules, CA) into
Agrobacterium tumefaciens strain GV3101, and
Arabidopsis plants (Ecotype Columbia) are transformed by infiltration (Bechtold,
et al., (1993)
C. R. Acad. Sci. Paris, 316: 1188-93). Seeds from the infiltrated plants are selected on germination medium
(Murashige-Skoog salts at 4.3 g/liter, MES at 0.5 g/liter, 1% sucrose, thiamine at
10 µg/liter, pyridoxine at 5 µg/liter, nicotinic acid at 5 µg/liter, myo-inositol
at 1 mg/liter, pH 5.8) containing Basta at 15 mg/liter.
Example 10: Antisense Inhibition of PBGD Using a GAL4/C1 Transactivator and a GAL4
Binding Site/Minimal CaMV 35S Promoter
[0130] Eighteen transgenic plants containing the GAL4 binding site/minimal CaMV 35S promoter/antisense
PBGD construct are transplanted to soil and grown to maturity in the greenhouse. Flowers
borne on the primary transformants are crossed to pollen from the homozygous GAL4/C1
transactivator line pAT53-103 (Guyer et al,
Genetics (1998) 149:633-649). F1 seeds are plated on MS + 2% sucrose medium (Murashige-Skoog
salts at 4.3 g/liter, Mes at 0.5 g/liter, 2% sucrose) eight lines segregate about
50% seedlings with a bleached lethal phenotype on plates. Thus, the inventors are
the first to demonstrate that the PBGD gene is essential for the growth of a plant.
Example 11: Isolation of a cDNA Encoding CPPO from Arabidopsis
[0131] Primer CR73 (5' TTG ACC CTT CCT TCT ATC CCC GAT TC 3': SEQ ID NO:30) is designed
to anneal to the complementary strand at 733-758 nucleotides from the 5' end of the
start codon of the predicted CPPO ORF from the BAC F21 B7 genomic sequence (GenBank
accession # AC002560), and primer CR75 (5' GTT GCC ATG CCT TGT GCT GCT CTG TA 3':
SEQ ID NO:31) is designed to anneal to the coding strand from 958-933 nucleotides
from the 5' end of the start codon of the predicted CPPO ORF from the BAC F21 B7 genomic
sequence (GenBank accession # AC002560). 3' RACE is performed using CR73 primer and
5' RACE is performed using CR75 primer with second strand cDNA from
Arabidopsis thaliana Ecotype Columbia as the template (Marathon cDNA Amplification Kit User Manual, Clontech).
The resulting PCR products are TA-ligated and cloned (Original TA Cloning Kit, Invitrogen),
and sequenced.
[0132] There are two differences between the sequence of the present invention and the genomic
sequence in the prior art. First, the genomic sequence contains GG at positions 67872-67873.
However, the inventors are the first to provide experimental evidence that the correct
sequence contains only one G at position 67872. In addition, the genomic DNA that
contains the CPPO ORF was not annotated correctly in the prior art with respect to
the number of exons and the exon boundaries, the inventors are the first to provide
experimental documentation of the correct ORF for the CPPO gene. The prior art indicates
these exon boundaries: 66178..66702, 66782..66857, 66946..67040, 67126..67209, 67391..67478,
67571..67695, 67801..67896. In the sequence of the present invention, base 66178 marks
the first base of the cDNA's start codon and base 68050 (using the numbering of the
deposited BAC which as indicated above is off by one nucleotide) marks the first base
of the cDNA's stop codon. The 3' end of the exon 7 is 67900 (using the numbering of
the deposited BAC which as indicated above is off by one nucleotide), and the 5' end
of the exon containing the stop codon (i.e., exon 8) is 67984 (using the numbering
of the deposited BAC which as indicated above is off by one nucleotide). The exon
boundaries for the cDNA disclosed herein are: 66178..66702, 66782..66857, 66946..67040,
67126..67209, 67391..67478, 67571..67695, 67801..67900, 67984..68301.
[0133] The
Arabidopsis cDNA sequence encoding the CPPO ORF is set forth in SEQ ID NO:9 and the encoded amino
acid sequence is set forth in SEQ ID NO:10.
Example 12: Construction of a Vector Containing a GAL4 Binding Site/Minimal 35S CaMV
Promoter Fused to Antisense CPPO
[0134] pJG304ΔXhol: Plasmid pJG304 (Guyer et al,
Genetics (1998), 149: 633-639) is partially digested with
Asp718 to isolate a full-length linear fragment. This fragment is ligated with a molar
excess of the 22 base oligonucleotide JG-L (5' GTA CCT CGA GTC TAG ACT CGA G 3'; SEQ
ID NO:32). Restriction analysis is used to identify a clone with this linker inserted
5' to the GAL4 DNA binding site, and this plasmid is designated pJG304ΔXhol.
[0135] pJG304/CO: Plasmid pJG304ΔXhol is digested with
Ncol and
Bg/II to excise the GUS gene. The GUS gene from pJG304ΔXhol is replaced with a CPPO
PCR product also digested with
Ncol and
Bg/II. This product is generated using primers CPPGO-F2 (5' CCC AGA TCT ATG GCT TCT
CAC TCG TCG 3'; SEQ ID NO:33) and CPPGO-R2 (5' CAT GCC ATG GTA TTC CCA TCT TGC TGA
AA 3'; SEQ ID NO:34) and the pFL61
Arabidopsis cDNA library (Minet et al. (1992) Plant J. 2: 417-422) as a template. Plasmid pJG304/CO
is constructed from the pJG304 digested vector ligated to the CPPO PCR product.
Example 13: Plant Transformation Vectors For CPPO Antisense Expression From The GAL4
Binding Site/CaMV Minimal 35S Promoter
[0136] pJG261/CO: pJG304/CO is cut with Xhol to excise the cassette containing the GAL4
DNA binding site/35S minimal promoter/antisense CPPO/CaMV terminator fusion. This
cassette is ligated into
XhoI-digested pJG261 (Guyer et al,
Genetics (1998), 149:633-639), producing pJG261/CO.
Example 14: Production Of GAL4 Binding Site/Minimal CaMV 35S Antisense CPPO Transgenic
Plants
[0137] pJG261/CO is electro-transformed (Bio-Rad Laboratories, Hercules, CA) into
Agrobacterium tumefaciens strain GV3101, and
Arabidopsis plants (Ecotype Columbia) are transformed by infiltration (Bechtold,
et al., (1993)
C. R. Acad. Sci. Paris, 316: 1188-93). Seeds from the infiltrated plants are selected on germination medium
(Murashige-Skoog salts at 4.3 g/liter, Mes at 0.5 g/liter, 1% sucrose, thiamine at
10 µg/liter, pyridoxine at 5 µg/liter, nicotinic acid at 5 µg/liter, myo-inositol
at 1 mg/liter, pH 5.8) containing Basta at 15 mg/liter.
Example 15: Antisense Inhibition of CPPO Using a GAL4/C1 Transactivator and a GAL4
Binding Site/Minimal CaMV 35S Promoter
[0138] Fifteen transgenic plants containing the GAL4 binding site/minimal CaMV 35S promoter/antisense
CPPO construct are transplanted to soil and grown to maturity in the greenhouse. Flowers
borne on the primary transformants are crossed to pollen from the homozygous GAL4/C1
transactivator line pAT53-103 (Guyer et al,
Genetics (1998) 149:633-649). F1 seeds are plated on MS + 2% sucrose medium (Murashige-Skoog
salts at 4.3 g/liter, Mes at 0.5 g/liter, 2% sucrose)13 lines segregate about 50%
seedlings with a bleached lethal phenotype on plates. Thus, the inventors are the
first to demonstrate that the CPPO gene is essential for the growth of a plant. Previously,
it has been shown that tobacco plants expressing a transgenic antisense construct
for this gene with 30-40% residual CPPO activity are sick (Kruse et al.
EMBO J (1995) 14: 3712-3720).
Example 16: Construction of a Vector Containing a GAL4 Binding Site/Minimal 35S CaMV
Promoter Fused to Antisense enoyl-ACP reductase (ENR-A)
[0139] pJG304/ENR-A: Plasmid pJG304ΔXhoI (Guyer et al,
Genetics (1998), 149: 633-639) is digested with
NcoI and
Bg/II to excise the GUS gene. The GUS gene from pJG304ΔXhoI is replaced with a ENR-A
PCR product digested
NcoI and
Bg/II. This product is generated using primers ENR-A-F2 (5'-CCC AGA TCT AAT GGC GGC
TAC AGC AGC TT-3'; SEQ ID NO:12) and ENR-A-R2 (5'-CAT GCC ATG GCT AAT TCT TGC TGT
TAA GG-3'; SEQ ID NO:13) and the pFL61
Arabidopsis cDNA library (Minet et al. (1992) Plant J. 2: 417-422) as a template. Plasmid pJG304/ENR-A
is constructed from the pJG304ΔXhoI digested vector ligated to the ENR-A PCR product.
Example 17: Plant Transformation Vectors For enoyl-ACP reductase (ENR-A) Antisense
Expression from the GAL4 Binding Site/CaMV Minimal 35S Promoter
[0140] pJG261/ENR-A: pJG304/ENR-A is cut with Xbal to excise the cassette containing the
GAL4 DNA binding site/35S minimal promoter/antisense enoyl-ACP reductase/CaMV terminator
fusion. This cassette is ligated into
SpeI-digested pJG261 (Guyer et al,
Genetics (1998), 149:633-639), producing pJG261/ENR-A.
Example 18: Production of GAL4 Binding Site/Minimal CaMV 35S Antisense ENR-A Transgenic
Plants
[0141] pJG261/ENR-A is electro-transformed (Bio-Rad Laboratories, Hercules, CA) into
Agrobacterium tumefaciens strain GV3101, and
Arabidopsis plants (Ecotype Columbia) are transformed by infiltration (Bechtold,
et al., (1993)
C. R. Acad. Sci. Paris, 316: 1188-93). Seeds from the infiltrated plants are selected on germination medium
(Murashige-Skoog salts at 4.3 g/liter, MES at 0.5 g/liter, 1% sucrose, thiamine at
10 µg/liter, pyridoxine at 5 µg/liter, nicotinic acid at 5 µg/liter, myo-inositol
at 1 mg/liter, pH 5.8) containing Basta at 15 mg/liter.
Example 19: Antisense Inhibition of ENR-A Using a GAL4/C1 Transactivator and a GAL4
Binding Site/Minimal CaMV 35S Promoter
[0142] Sixteen transgenic plants containing the GAL4 binding site/minimal CaMV 35S promoter/antisense
enoyl-ACP reductase construct are transplanted to soil and grown to maturity in the
greenhouse. Flowers borne on the primary transformants are crossed to pollen from
the homozygous GAL4/C1 transactivator line pAT53-103 (Guyer et al,
Genetics (1998) 149:633-649). F1 seeds are plated on MS + 2% sucrose medium (Murashige-Skoog
salts at 4.3 g/liter, Mes at 0.5 g/liter, 2% sucrose). Two lines segregate about 50%
seedling with a bleached phenotype on plates. These affected seedlings die shortly
after transplanting to soil. Thus, the inventors are the first to demonstrate that
the ENR-A gene is essential for the growth of a plant.
Example 20a: Expression of Recombinant CBL Protein in E. coli
[0143] The coding region of the protein, corresponding to the cDNA clone SEQ ID NO:3, is
subcloned into an appropriate expression vector, and transformed into
E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBluescript
(Stratagene, La Jolla, CA; USA), pFLAG (International Biotechnologies, Inc., New Haven,
CT, USA), and pTrcHis (Invitrogen, La Jolla, CA, USA).
E. coli is cultured, and expression of CBL activity is confirmed. Protein conferring CBL
activity is isolated using standard techniques.
Example 20b: Expression of Recombinant UROD Protein in E. coli
[0144] The coding region of the protein, corresponding to the cDNA clone SEQ ID NO:5, is
subcloned into an appropriate expression vector, and transformed into
E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBluescript
(Stratagene, La Jolla, CA, USA), pFLAG (International Biotechnologies, Inc., New Haven,
CT, USA), and pTrcHis (Invitrogen, La Jolla, CA, USA).
E. coli is cultured, and expression of UROD activity is confirmed. Protein conferring UROD
activity is isolated using standard techniques.
Example 20c: Expression of Recombinant PBGD Protein in E. coli
[0145] The coding region of the protein, corresponding to the cDNA clone SEQ ID NO:7, is
subcloned into an appropriate expression vector, and transformed into
E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBluescript
(Stratagene, La Jolla, CA, USA), pFLAG (International Biotechnologies, Inc., New Haven,
CT, USA), and pTrcHis (Invitrogen, La Jolla, CA, USA).
E. coli is cultured, and expression of PBGD activity is confirmed. Protein conferring PBGD
activity is isolated using standard techniques.
Example 20d: Expression of Recombinant CPPO Protein in E. coli
[0146] The coding region of the protein, corresponding to the cDNA clone SEQ ID NO:9, is
subcloned into an appropriate expression vector, and transformed into
E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBluescript
(Stratagene, La Jolla, CA, USA), pFLAG (International Biotechnologies, Inc., New Haven,
CT, USA), and pTrcHis (Invitrogen, La Jolla, CA, USA).
E. coli is cultured, and expression of CPPO activity is confirmed. Protein conferring CPPO
activity is isolated using standard techniques.
Example 20e: Expression of Recombinant ENR-A Protein in E. coli
[0147] The coding region of the protein, corresponding to the cDNA clone SEQ ID NO:1, is
subcloned into an appropriate expression vector, and transformed into
E. coli using the manufacturer's conditions. Specific examples include plasmids such as pBluescript
(Stratagene, La Jolla, CA, USA), pFLAG (International Biotechnologies, Inc., New Haven,
CT, USA), and pTrcHis (Invitrogen, La Jolla, CA, USA).
E. coli is cultured, and expression of ENR-A activity is confirmed. Protein conferring ENR-A
activity is isolated using standard techniques.
Example 21: In vitro Recombination of ENR-A Genes by DNA Shuffling
[0148] The nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or
SEQ ID NO:9, respectively, is amplified by PCR. The resulting DNA fragment is digested
by DNasel treatment essentially as described (Stemmer et al. (1994)
PNAS 91: 10747-10751) and the PCR primers are removed from the reaction mixture. A PCR
reaction is carried out without primers and is followed by a PCR reaction with the
primers, both as described (Stemmer et al. (1994)
PNAS 91: 10747-10751). The resulting DNA fragments are cloned into pTRC99a (Pharmacia,
Cat no: 27-5007-01) for use in bacteria, and transformed into a bacterial strain deficient
in ENR-A, CBL, UROD, PBGD, or CPPO activity , respectively, by electroporation using
the Biorad Gene Pulser and the manufacturer's conditions. The transformed bacteria
are grown on medium that contains inhibitory concentrations of an inhibitor of ENR-A,
CBL, UROD, PBGD, or CPPO activity, respectively, and those colonies that grow in the
presence of the inhibitor are selected. Colonies that grow in the presence of normally
inhibitory concentrations of inhibitor are picked and purified by repeated restreaking.
Their plasmids are purified and the DNA sequences of cDNA inserts from plasmids that
pass this test are then determined. Alternatively, the DNA fragments are cloned into
expression vectors for transient or stable transformation into plant cells, which
are screened for differential survival and/or growth in the presence of an inhibitor
of ENR-A, CBL, UROD, PBGD, or CPPO activity, respectively. In a similar reaction,
PCR-amplified DNA fragments comprising the
Arabidopsis ENR-A, CBL, UROD, PBGD, or CPPO gene, respectively, encoding the protein and PCR-amplified
DNA fragments derived from or comprising another
ENR-A, CBL, UROD, PBGD, or CPPO gene, respectively, are recombined
in vitro and resulting variants with improved tolerance to the inhibitor are recovered as
described above.
Example 22a: In vitro Recombination of CBL Genes by Staggered Extension Process
[0149] The
Arabidopsis CBL gene and another CBL gene, or homologs thereof, or fragments thereof, are each
cloned into the polylinker of a pBluescript vector. A PCR reaction is carried out
essentially as described (Zhao et al. (1998)
Nature Biotechnology 16: 258-261) using the "reverse primer" and the "M13 -20 primer" (Stratagene Catalog).
Amplified PCR fragments are digested with appropriate restriction enzymes and cloned
into pTRC99a and mutated CBL genes are screened as described in Example 21.
Example 22b: In vitro Recombination of UROD Genes by Staggered Extension Process
[0150] The
Arabidopsis UROD gene and another UROD gene, or homologs thereof, or fragments thereof, are each
cloned into the polylinker of a pBluescript vector. A PCR reaction is carried out
essentially as described (Zhao et al. (1998)
Nature Biotechnology 16: 258-261) using the "reverse primer" and the "M13 -20 primer" (Stratagene Catalog).
Amplified PCR fragments are digested with appropriate restriction enzymes and cloned
into pTRC99a and mutated UROD genes are screened as described in Example 21.
Example 22c: In vitro Recombination of PBGD Genes by Staggered Extension Process
[0151] The
Arabidopsis PBGD gene and another PBGD gene, or homologs thereof, or fragments thereof, are each
cloned into the polylinker of a pBluescript vector. A PCR reaction is carried out
essentially as described (Zhao et al. (1998)
Nature Biotechnology 16: 258-261) using the "reverse primer" and the "M13 -20 primer" (Stratagene Catalog).
Amplified PCR fragments are digested with appropriate restriction enzymes and cloned
into pTRC99a and mutated PBGD genes are screened as described in Example 21.
Example 22d: In vitro Recombination of CPPO Genes by Staggered Extension Process
[0152] The
Arabidopsis CPPO gene and another CPPO gene, or homologs thereof, or fragments thereof, are each
cloned into the polylinker of a pBluescript vector. A PCR reaction is carried out
essentially as described (Zhao et al. (1998)
Nature Biotechnology 16: 258-261) using the "reverse primer" and the "M13 -20 primer" (Stratagene Catalog).
Amplified PCR fragments are digested with appropriate restriction enzymes and cloned
into pTRC99a and mutated CPPO genes are screened as described in Example 21.
Example 22e: In vitro Recombination of ENR-A Genes by Staggered Extension Process
[0153] The
Arabidopsis ENR-A gene and another ENR-A gene, or homologs thereof, or fragments thereof, are
each cloned into the polylinker of a pBluescript vector. A PCR reaction is carried
out essentially as described (Zhao et al. (1998)
Nature Biotechnology 16: 258-261) using the "reverse primer" and the "M13 -20 primer" (Stratagene Catalog).
Amplified PCR fragments are digested with appropriate restriction enzymes and cloned
into pTRC99a and mutated ENR-A genes are screened as described in Example 21.
Example 23: In Vitro Binding Assays
[0154] Recombinant ENR-A, CBL, UROD, PBGD, or CPPO protein, respectively, is obtained, for
example, according to Example 20. The protein is immobilized on chips appropriate
for ligand binding assays using techniques which are well known in the art. The protein
immobilized on the chip is exposed to sample compound in solution according to methods
well know in the art. While the sample compound is in contact with the immobilized
protein measurements capable of detecting protein-ligand interactions are conducted.
Examples of such measurements are SELDI, biacore and FCS, described above. Compounds
found to bind the protein are readily discovered in this fashion and are subjected
to further characterization.
[0155] Various modifications of the invention described herein will become apparent to those
skilled in the art. Such modifications are intended to fall within the scope of the
appended claims.
Example 24: CBL Activity Assay
[0156] The CBL activity assay is derived from Stintjes
et al. (1992)
Anal. Biochem. 206, 334-343. The reaction volumes are preferably the ones described below, but can
be varied depending on the experimental requirements. 0.01-1.0 x 10
-3 unit of an enzyme having CBL activity (one unit of activity is defined as the amount
of enzyme required to produce 1 mmol/min of product) and 0.5-5 mM, but preferably
1 mM L(+)cystathionine (cyn) are mixed in a final volume of 10 mL 10 mM Tris-HCl (pH
7.0-9.0, but preferably 8.5) and 1-20 mM, but preferably 10 mM pyridoxal 5'-phosphate.
The production of pyruvate is determined preferably according to Stintjes
et al. (1992)
Anal. Biochem. 206, 334-343 by adding 5 mL of 20 mM o-phenylenediamine in 0.6 M hydrochloric acid.
Fluorescence intensity is measured for the solution with an excitation wavelength
of 410 ± 10 nm and an emission wavelength of 535 ± 10 nm. Alternatively, the absorbance
of the solution may be measured with a wavelength of 410 ± 10 nm.
[0157] Alternatively, pyruvate formation is quantified by a coupled reaction procedure.
In this case, 0.5 units of lactate dehydrogenase and 0.2 mM NADH are added and the
fluorescence intensity of the solution is measured with an excitation wavelength of
340 ± 10 nm and an emission wavelength of 410 ± 10 nm. Alternatively, the absorbance
of the solution may be measured at 340 nm. Other ways to measure the activity of this
enzyme known in the art may be used.
Example 25: In Vitro Functional Assay for PBGD Activity
[0158] Recombinant PBGD protein is obtained, for example, according to Example 5. The protein
can be used in a functional PBGD activity assay as described in Jones and Leadbeater
(1997)
Meth. Enz. 281, 327-336. The reaction volumes are preferably the ones described below, but can
be varied depending on the experimental requirements. 0.01-1.0 x 10
-3 unit of an enzyme having PBGD activity (one unit of activity is defined as the amount
of enzyme required to produce 1 mmol/min of product) and 0.01-5 mM, but preferably
0.05 mM, porphobilinogen are mixed in a final volume of 10 mL 20 mM Tris-HCl (pH 7.0-9.0,
but preferably 8.0) and 0.1-10 mM, but preferably 2 mM, dithiothreitol. The production
of hydroxymethylbilane is determined indirectly preferably according to Jones and
Leadbeater (1997)
Meth. Enz. 281, 327-336 by adding 5 mL of 5 mM hydrochloric acid followed by 5 mL of 0.1 % benzoquinone
in methanol. Fluorescence intensity is measured for the solution with an excitation
wavelength of 405 ± 10 nm and an emission wavelength of 620 ± 10 nm.
Example 26: In vitro Enzymatic Assay for CPPO Activity
[0159] Recombinant CPPO protein is obtained, for example, according to Example 6. The protein
is used for
in vitro enzymatic assays. At least three procedures are used by one skilled in the art. First,
CPPO is combined with a protoporphyrinogen oxidase. In this procedure, coproporphyrinogen
III is converted to protoporphyrinogen IX by CPPO and protoporphyrinogen IX is converted
to protoporphyrin IX by protoporphyrinogen oxidase (Labbe, Camadro, and Chambon (1985)
Anal. Biochem., 149: 248-260). The formation of protoporphyrin IX is measured colorimetrically
or fluorimetrically. Alternatively, CPPO is assayed singularly by converting protoporphyrinogen
IX, the product of the CPPO enzymatic activity, to protoporphyrin IX chemically using
an oxidizing agent known to one skilled in the art (Yoshinga (1997) Meth. Enz. 281:
355-367). The formation of protoporphyrin IX can be measured colorimetrically or fluorimetrically.
Additionally, the formation of protoporphyrinogen IX from coproporphyrinogen III is
measured by HPLC (Rossi, Garcia-Webb, and Costin (1989) Clin. Chim. Acta 181: 115-117).
Example 27: Plastid Transformation
Transformation vectors
[0160] For expression of a nucleotide sequence encoding a polypeptide having ENR-A, CBL,
UROD, PBGD, or CPPO activity, respectively, encoding in plant plastids, plastid transformation
vector pPH143 or pPH145 (WO 97/32011) is used; and this reference is incorporated
herein by reference. The nucleotide sequence is inserted into pPH143 thereby replacing
the PROTOX coding sequence. This vector is then used for plastid transformation and
selection of transformants for spectinomycin resistance. Alternatively, the nucleotide
sequence is inserted in pPH143 so that it replaces the
aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.
Plastid Transformation
[0161] Seeds of
Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in a 1" circular array on T agar
medium and bombarded 12-14 days after sowing with 1 µm tungsten particles (M10, Biorad,
Hercules, CA) coated with DNA from plasmids pPH143 and pPH145 essentially as described
(Svab, Z. and Maliga, P. (1993)
Proc. Natl. Acad. Sci. USA 90, 913-917). Bombarded seedlings are incubated on T medium for two days after which
leaves are excised and placed abaxial side up in bright light (350-500 µmol photons/m
2/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990)
Proc. Natl. Acad. Sci. USA 87, 8526-8530) containing 500 µg/ml spectinomycin dihydrochloride (Sigma, St. Louis,
MO). Resistant shoots appearing underneath the bleached leaves three to eight weeks
after bombardment are subcloned onto the same selective medium, allowed to form callus,
and secondary shoots isolated and subcloned. Complete segregation of transformed plastid
genome copies (homoplasmicity) in independent subclones is assessed by standard techniques
of Southern blotting (Sambrook et al., (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). Homoplasmic shoots are rooted
aseptically on spectinomycin-containing MS/IBA medium (McBride, K. E. et al. (1994)
Proc. Natl. Acad. Sci. USA 91, 7301-7305) and transferred to the greenhouse.